CN111228222A

CN111228222A - Nano bowl-supported drug-loaded liposome and preparation method and application thereof

Info

Publication number: CN111228222A
Application number: CN202010145448.8A
Authority: CN
Inventors: 方超; 陈中坚; 杨思聪
Original assignee: Shanghai Jiaotong University School of Medicine
Current assignee: Shanghai Eryi Investment Management Co.,Ltd.; Shanghai Jiaotong University School of Medicine
Priority date: 2020-03-03
Filing date: 2020-03-03
Publication date: 2020-06-05
Anticipated expiration: 2040-03-03
Also published as: WO2021174894A1; US20230052618A1; CN111228222B

Abstract

The invention relates to the technical field of medicines, in particular to a nano bowl-supported drug-loaded liposome and a preparation method and application thereof, wherein the preparation method of the nano bowl-supported drug-loaded liposome comprises the following steps: and (3) carrying the medicine successfully by carrying out incubation ultrasound on the nano bowl and the liposome and using an ammonium sulfate active medicine carrying method to obtain the nano bowl supported medicine carrying liposome. The invention also comprises application of the nanometer bowl-supported drug-loaded liposome in preparation of antitumor drugs. Its advantages are: the drug-loaded liposome supported by the nano bowl (nanobowl) can resist the influence of plasma protein and blood flow shearing force on drug leakage, improve the transfer of the drug at tumor parts, improve the stability of a carrier and improve the anti-tumor curative effect. The physical support is designed for the cavity of the all-water nano liposome, and the application prospect is good.

Description

Nano bowl-supported drug-loaded liposome and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a nano bowl-supported drug-loaded liposome and a preparation method and application thereof.

Background

Cancer, i.e., malignant tumor, has now become the second leading cause of death in the world, and seriously threatens the life and health of human beings. The latest published data of the world health organization in 2/3.2017 shows that 880 ten thousand people die of cancer each year around the world, accounting for nearly one sixth of the total number of death each year around the world, and 1400 ten thousand new cancer cases each year, and the number is expected to increase to 2100 ten thousand by 2030. How to overcome the worldwide problem of cancer has become a hot issue which is urgently desired to be solved in the field of life science.

However, the therapeutic effect of cancer drugs is greatly limited due to their toxicity to normal tissue cells and their extremely fast clearance rate. Due to the generation of the nano-carrier technology, the combination of the nano-carrier technology and cancer treatment can break through the limitation of the factors of the medicine and exert better curative effect because of good biocompatibility, ideal long circulation time and accurate targeting.

One important mechanism of action of nano-drugs, which can not be achieved by such single drugs, is Enhanced Permeability and Retention (EPR) effect. The EPR effect refers to the phenomenon that some macromolecular substances with specific sizes (such as liposome, nanoparticle and some macromolecular drugs) are easier to permeate into tumor tissues and stay for a long time (compared with normal tissues). The nano-drug treatment strategy based on the EPR effect theory is that the drug peak concentration (Cmax) is maximized by changing the pharmacokinetics and biodistribution of the drug, and the area under the drug concentration-time relationship curve (AUC) of the drug at plasma and tumor tissues is increased, so that the drug curative effect and the organism tolerance are improved, and the drug treatment level of the drug at a target part is prolonged.

Among these, liposomes, the first nanoparticle approved for anti-tumor therapy, together with the rest of the nanoparticles designed on the basis of liposomes, account for a considerable proportion of clinical nanotherapeutics. However, while encapsulation of chemotherapeutic drugs in liposomes can improve the pharmacokinetics and biological tissue distribution of the drug, the currently marketed liposomal drug formulations do not significantly improve the overall survival rate over traditional chemotherapeutic drugs. As can be seen, liposome delivery systems still face difficulties and challenges.

Among the challenges, liposome stability appears to be critical, which determines the changes in drug loading, leakage rate, and release rate of the drug during preparation, storage, and in vivo metabolism of the drug-loaded liposomes. The main component of the liposome is phospholipid, and a part of phospholipid contains unsaturated fatty acid branched chains, so that the phospholipid is easy to oxidize and hydrolyze, and the mobility of phospholipid bilayers is reduced, the permeability is increased, the agglomeration phenomenon is aggravated, the leakage rate of the medicament is increased, the medicament loading rate is reduced, and the like. Research shows that the properties of the liposome formed by different phospholipid components can be changed correspondingly, thereby influencing the stability and the drug release rate of the liposome. The phase transition temperature, as another important factor, affects the stability of liposomes for storage. When the temperature exceeds the phase transition temperature of the phospholipid, the phospholipid is converted from a gel state to a liquid crystal state, so that the lipid bilayer structure is loose, the stability is reduced, the drug is released, and the storage period is shortened. In addition, the thermosensitive liposome is based on the principle of phase transition temperature, and the purpose of controlling drug release is achieved by changing the temperature.

The stability of liposomes in vivo is undoubtedly the key point in determining the therapeutic effect of drugs. Clearly, the theory of the EPR effect alone may not fully explain the changes that occur in liposomes in the body. Because many biological processes, such as the hydrodynamic action of the blood circulation system, the interaction with various proteins and cytokines, the tissue penetration action of the nanoparticles, etc., are required before the nanoparticles reach the tumor tissue after intravenous injection. The change of the shearing force in the blood flow can change the deformation degree of the liposome and influence the stability of the liposome; research indicates that high-density lipoprotein in serum can be combined with phospholipid to form a hole gap; the liposome can activate a complement system, and a hydrophilic channel is formed on the surface of the liposome, so that the permeability of the liposome is increased, and the content is leaked in a large amount; serum albumin can even be combined with the liposome to form a crown structure, which affects the stability of the liposome; meanwhile, the mononuclear phagocyte system in the circulatory system can quickly recognize and remove the liposome. Therefore, how to improve the composition, form, size, Zeta potential and surface characteristics of the liposome to overcome the above difficulties and better exert the therapeutic effect of the liposome drug is important.

The invention aims to establish a novel liposome drug delivery system, namely a nano bowl-supported adriamycin liposome, based on the challenges faced by the liposome drug preparation at the present stage. Due to the unique morphological structure of the nano bowl, enough lumen space is reserved for the successful loading of adriamycin while the liposome is provided with enough rigid supporting effect. Through years of clinical application, doxorubicin liposome, which is the nano-drug on the market at the earliest, has certain improvement on the treatment of tumor patients, but also has some defects of the doxorubicin liposome. The addition of the nanometer bowl is expected to solve certain practical problems, and has extremely high scientific value and potential clinical transformation significance. According to the invention, different effects of the nano bowl-supported adriamycin liposome on tumor tissues and normal tissues are considered, so that the in-vivo anti-tumor curative effect of the nano bowl-supported adriamycin liposome is comprehensively evaluated.

The nano bowl-supported drug-loaded liposome and the preparation method and application thereof are not reported at present.

Disclosure of Invention

The first purpose of the invention is to provide a nano bowl supported drug-loaded liposome aiming at the defects of the prior art.

The second purpose of the present invention is to provide a preparation method of the nanometer bowl-supported drug-loaded liposome as described above, aiming at the defects of the prior art.

The third purpose of the present invention is to provide the use of the nanometer bowl supporting drug-loaded liposome as described above, aiming at the defects of the prior art.

The fourth purpose of the invention is to provide a nano bowl-supported adriamycin/irinotecan/vincristine liposome aiming at the defects of the prior art.

The fourth purpose of the present invention is to provide a preparation method of the nanometer bowl-supported adriamycin/irinotecan/vincristine liposome described above, aiming at the defects of the prior art.

In order to achieve the first purpose, the invention adopts the technical scheme that:

the preparation method of the nano bowl supported drug-loaded liposome comprises the following steps:

(1) preparing a nanometer bowl: preparing polystyrene nano-particles → preparing MPS coated modified polystyrene nano-particles → preparing peanut-shaped nano-particles → preparing silica modified peanut-shaped nano-particles → obtaining a nano bowl;

(2) preparing a nano bowl supported no-load liposome: carrying out amination modification on APTES and the nano bowl prepared in the step (1) to obtain an aminated nano bowl → transferring the aminated nano bowl into a prepared ammonium sulfate solution by a centrifugal method to obtain an aminated ammonium sulfate solution of the nano bowl, oscillating the ammonium sulfate solution to form a vesicle coated with the nano bowl → carrying out probe ultrasound on the vesicle coated with the nano bowl to obtain a nano bowl supported no-load liposome;

(3) preparing a nano bowl supported drug-loaded liposome: the drug is encapsulated by using an ammonium sulfate active drug loading method to obtain the nanometer bowl supported drug-loaded liposome.

Preferably, the drug in step (3) is selected from any one of adriamycin, irinotecan and vincristine.

In order to achieve the second object, the invention adopts the technical scheme that:

the preparation method of the nanometer bowl supported drug-loaded liposome comprises the following steps:

In order to achieve the third object, the invention adopts the technical scheme that:

the nanometer bowl supported drug-loaded liposome is applied to the preparation of anti-tumor drugs.

In order to achieve the fourth object, the invention adopts the technical scheme that:

the preparation method of the nano bowl supported adriamycin/irinotecan/vincristine liposome comprises the following steps:

(3) preparation of nano bowl supported adriamycin/irinotecan/vincristine liposome: by utilizing an ammonium sulfate active drug loading method, the adriamycin/irinotecan/vincristine is entrapped to obtain the nano bowl-supported adriamycin/irinotecan/vincristine liposome.

Preferably, the polystyrene nanoparticles in the step (1) have a particle size of 45-55 nm; the peanut-shaped nano particles are prepared from styrene monomers and MPS modified polystyrene nano particles according to the ratio of Vstyrene to VMPSNPs of 3: 1; the silica-modified peanut-like nanoparticles were prepared with 0.3g TEOS.

Preferably, the nano bowl-supported adriamycin/irinotecan/vincristine liposome is characterized in that the average particle size is 140-150nm, and the Zeta potential is-18 to-16 mV.

In order to achieve the fifth object, the invention adopts the technical scheme that:

the preparation method of the nano bowl supported adriamycin/irinotecan/vincristine liposome comprises the following steps: (1) preparing a nanometer bowl: preparing polystyrene nano-particles → preparing MPS coated modified polystyrene nano-particles → preparing peanut-shaped nano-particles → preparing silica modified peanut-shaped nano-particles → obtaining a nano bowl;

Preferably, the preparation method of the nanometer bowl in the step (1) comprises the following steps:

1) preparing polystyrene nano-particles: synthesizing polystyrene nanoparticles by using an emulsion polymerization method by using styrene as a monomer, SDS as an emulsifier and KPS as an initiator;

2) preparing MPS coated modified polystyrene nanoparticles: adding styrene, MPS and AIBN on the basis of the synthesized polystyrene nano-particles, and synthesizing MPS coated modified polystyrene nano-particles through polymerization reaction;

3) preparing peanut-shaped nanoparticles: mixing the MPS coated modified polystyrene nano-particles prepared in the step 2) with styrene and VBS, putting the mixture into ultrapure water for stirring, utilizing the swelling effect of the polystyrene to enable the MPS coated modified polystyrene nano-particles to deform, expand and break, then adding AIBN, and initiating polymerization reaction again to finally form peanut-shaped nano-particles;

4) preparing the silica modified peanut-shaped nanoparticles: transferring the peanut-shaped nanoparticles obtained in the step 3) to absolute ethyl alcohol in a manner of ultracentrifugation and redispersion, then adding 25% concentrated ammonia water to prepare an ethanol solution containing 50% TEOS, and slowly dropwise adding to obtain the silica-modified peanut-shaped nanoparticles;

5) preparing a nano bowl: transferring the silica modified peanut-shaped nanoparticles obtained in the step 4) to a rotary evaporator, volatilizing excessive ethanol, adding tetrahydrofuran for dissolving, and performing ultracentrifugation to collect precipitates to obtain a final product, namely a nano bowl.

Preferably, the particle size of the polystyrene nanoparticles in the step (1) is 50 nm; the peanut-shaped nano particles are polystyrene nano particles modified by styrene monomers and MPS according to V_styrene:V_MPSNPsPrepared as 3: 1; the silica-modified peanut-like nanoparticles were prepared with 0.3g TEOS.

The prepared adriamycin liposome supported by the nanometer bowl provides a hard inner container for the liposome through the supporting effect of the nanometer bowl, so that the adriamycin liposome can bear the impact and damage of various factors in a circulating system, the leakage of content drugs is reduced as far as possible before reaching a tumor part, more adriamycin reaches the tumor part along the circulating system, the stability of liposome adriamycin (DOX) for active drug loading is improved, and the curative effect of drugs is better exerted.

The invention has the advantages that:

1. the nano bowl (nanobowl) supported drug-loaded liposome prepared by the invention can resist the influence of plasma protein and blood flow shearing force on drug leakage. The method improves the delivery of the drug at the tumor site, and improves the anti-tumor effect. Compared with other methods for improving the stability by changing liposome double layers, composition components and the like, the method designs physical support for the cavity of the all-water nanoliposome. The nanometer bowl stabilizes the liposome, and improves the stability of the carrier and the drug release.

2. The invention overcomes the defects of the prior art (the liposome drug delivery for cancer treatment is possibly limited due to the leakage of the drug in blood circulation), improves the stability of the liposome drug for active drug loading by embedding a hard nano bowl in a liposome water cavity, optimizes the types of raw materials and the proportion and process parameters among the raw materials, obtains the nano bowl supported drug-loaded liposome with the best curative effect, improves the delivery of the drug at a tumor part by the nano bowl supported drug-loaded liposome which can resist the influence of plasma protein and blood flow shearing force on drug leakage, improves the anti-tumor curative effect without toxic and side effects, designs physical support for a full-water nano liposome cavity compared with other methods for improving the stability by changing a liposome double layer, components and the like, improves the survival rate of breast cancer patients, lightens the economic burden of the patients, has good application prospect.

Drawings

FIG. 1 is a schematic diagram of a nano bowl synthesis circuit.

FIG. 2 is a drawing: (A-E) are DLS particle size distribution maps of polystyrene nanoparticles, MPS-coated modified polystyrene nanoparticles, peanut-shaped nanoparticles, silica-modified peanut-shaped nanoparticles and a nanometer bowl respectively; (F-J) are Zeta potential distribution diagrams corresponding to polystyrene nanoparticles, MPS modified polystyrene nanoparticles, peanut-shaped nanoparticles, silica modified peanut-shaped nanoparticles and a nanometer bowl, respectively.

FIG. 3 is a schematic representation of: (A) transmission electron micrographs of polystyrene nanoparticles, (B) peanut-like nanoparticles, and (C) silica-modified peanut-like nanoparticles.

FIG. 4 is a drawing of: the (A, B) is a transmission electron microscope image of the nanometer bowl, (C) is the opening angle of the nanometer bowl estimated according to the radius and the length-width ratio, and (D-G) are the nanometer bowls with different angles under the transmission electron microscope.

FIG. 5 is a schematic representation of: (A) the transmission electron microscope images of the small-size polystyrene nanoparticles are (B, C) the particle size distribution and Zeta potential diagram of the small-size polystyrene nanoparticles.

FIG. 6 is a schematic representation of: (A) the transmission electron microscope images of peanut-shaped nanoparticles synthesized at a feed ratio of 3:1, and (B) the transmission electron microscope images of peanut-shaped nanoparticles synthesized at a feed ratio of 9: 1.

FIG. 7 is a drawing of: (A) the transmission electron microscope image of the nano bowl synthesized with 0.3g of TEOS is shown, and the transmission electron microscope image of the nano bowl synthesized with 0.5g of TEOS is shown.

FIG. 8 is a schematic diagram of a nano bowl supported doxorubicin liposome synthesis circuit.

FIG. 9 is a drawing of: (A) the transmission electron microscope images of the aminated nanometer bowl are shown, and the (B, C) are respectively the particle size distribution and the Zeta potential diagram of the aminated nanometer bowl.

FIG. 10 is a drawing of: (A) is a nano bowl supported liposome mode diagram, (B) is a transmission electron microscope diagram of the nano bowl supported liposome, (C, D, E and F) are respectively a particle size distribution and a Zeta potential diagram of the nano bowl supported liposome and a common liposome.

FIG. 11 is a drawing of: (A) the ultraviolet absorption and fluorescence spectrum of doxorubicin hydrochloride is shown, and the ultraviolet absorption and fluorescence spectrum of a DiR probe is shown in (B).

FIG. 12 is a drawing of: (A) the ultraviolet absorption and fluorescence spectrum of the adriamycin liposome supported by the nanometer bowl is shown, and the ultraviolet absorption and fluorescence spectrum of the common adriamycin liposome is shown in (B).

Figure 13 is a schematic diagram of active loading of ammonium sulfate.

FIG. 14 is a drawing of: (A) the drug leakage rate of the common adriamycin liposome and the nano bowl-supported adriamycin liposome in the whole serum is reduced; (B) the nanometer bowl supports the liposome, the particle size and the Zeta potential of the liposome are changed within 24h in serum, and n is 3.

FIG. 15 is a drawing of: the (A, D) is the state of redispersion after the nanometer bowl-supported adriamycin liposome and the common adriamycin liposome are frozen and dried respectively, and the (B, C, E, F) is a particle size distribution comparison chart before and after the nanometer bowl-supported adriamycin liposome and the common adriamycin liposome are frozen and dried respectively.

FIG. 16 is a graph of particle size distribution and Zeta potential change of nano bowl-supported doxorubicin liposome during storage at 4 deg.C, where n is 3.

FIG. 17 is a drawing of: (A) the condition that the tumor cells shoot under a laser confocal microscope take up the nanoparticles, wherein Bar is 25 m; (B) the fluorescence intensity of the nanoparticles taken up by the cells was quantitatively analyzed, and the result was expressed as "mean ± SD", and n ═ 5. P <0.05, P <0.01, P < 0.001.

FIG. 18 is a drawing of: (A) effect of free doxorubicin hydrochloride, ordinary doxorubicin liposomes, nano-bowl supported doxorubicin liposomes on viability of 4T1 breast cancer cells, (B) effect of empty nano-bowl supported liposomes without drug loading on viability of 4T1 cells, results are expressed as mean ± SD, and n ═ 4. P <0.01, P < 0.001.

FIG. 19 is a graph showing that nano bowl supported doxorubicin liposomes have good therapeutic effect on BALB/c female mouse with 4T 1-bearing breast cancer; (A) a schematic administration diagram; (B, C) tumor volume and mouse weight of tumor-bearing mice of each administration group respectively, wherein n is 8; in the B picture, a is the statistical difference between the tumor volumes of free adriamycin and normal saline and blank carrier group mice, B is the statistical difference between the tumor volumes of common adriamycin liposome and normal saline and blank carrier group mice, and c is the statistical difference between the tumor volumes of nano bowl-supported adriamycin liposome and the tumor volumes of the rest four groups of experimental group mice; (D) survival curves for tumor-bearing mice of each dosing group, P <0.05, P <0.01, P < 0.001.

FIG. 20 is a (A) pathological section and immunohistochemical staining analysis of tumor tissue of 4T1 tumor-bearing mice, wherein Bar is 100m, and (B, C) are statistical graphs of the positive rates of TUNEL and PCNA in tumor tissue after treatment of each experimental group; the results are expressed as mean ± SD, n ═ 5,. P <0.05,. P <0.01,. P < 0.001.

FIG. 21 shows immunohistochemical staining analysis of important organ tissues of 4T1 tumor-bearing mice, Bar 100 m.

FIG. 22 is a drawing of: (A) the drug leakage stability of the common irinotecan liposome and the nano bowl-supported irinotecan liposome and (B) the common vincristine liposome and the nano bowl-supported vincristine liposome in FBS.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

The abbreviations used in the present invention are shown in Table 1.

TABLE 1 abbreviation List

Example 1 construction and characterization of nanometer bowls

Construction of one, nanometer bowl

1 instruments and materials

1.1 instrumentation

TABLE 2

1.2 materials and reagents

TABLE 3

Styrene (meth) acrylic acid ester	Sigma Co USA
		SDS	SANGON BIOTECH (SHANGHAI) Co.,Ltd.
KPS	Shanghai Aladdin Chemicals Ltd
		MPS	Sigma Co USA
AIBN	Shanghai Aladdin Chemicals Ltd
		VBS(90％)	Sigma Co USA
TEOS	Sigma Co USA
		Anhydrous ethanol	Chemical reagents of Chinese medicine Co Ltd
25% concentrated ammonia water	Shanghai Aladdin Chemicals Ltd
		Tetrahydrofuran (THF)	Chemical reagents of Chinese medicine Co Ltd

2 method of experiment

2.1 polystyrene nanoparticle Synthesis

The polystyrene nano-particles with the size of 50nm are synthesized by adopting an emulsion polymerization method. The method comprises the following specific steps:

1) precisely weighing 1.00g of sodium dodecyl sulfate and placing the sodium dodecyl sulfate in a clean 250mL three-neck flask;

2) precisely measuring 85mL of ultrapure water by using a 100mL measuring cylinder, and pouring the ultrapure water into a 250mL three-neck flask filled with sodium dodecyl sulfate;

3) three bottle mouths of the three-necked flask are respectively connected with a spherical condenser tube and two rubber plugs;

4) inserting a needle head into a rubber plug of one bottle mouth of the three-neck flask, completely submerging the needle head to a position below the liquid level, simultaneously connecting a guide pipe to the other end of the needle head, and communicating argon to the other end of the guide pipe;

5) opening an argon steel bottle valve, introducing argon into the three-neck flask, adjusting the valve, controlling the flow rate of the argon, and observing that bubbles are uniformly formed in the liquid level of the three-neck flask;

6) meanwhile, stirring is kept for 1h at the rotating speed of 250rpm by an HS-70 type constant-temperature magnetic stirrer;

7) taking down the needle head on the three-neck flask, and switching the guide pipe which is communicated with the argon gas to be communicated with the spherical condenser pipe to keep the ventilation;

8) precisely transferring 20.00g of styrene monomer, inserting a rubber plug into a 50mL injector, injecting the styrene monomer into a three-neck flask, and continuously keeping magnetic stirring for 30 min;

9) opening a temperature rise switch of the constant-temperature magnetic stirrer, and adjusting the temperature to heat the reaction system to 70 ℃;

10) precisely weighing 0.10g of potassium persulfate, adding into 10mL of ultrapure water, and vortexing by using an MS2 type vortex mixer for 1min to fully dissolve the potassium persulfate;

11) after the temperature of the reaction flask is balanced, slowly dropwise adding a potassium persulfate solution prepared in advance by using a 10mL syringe;

12) under the protection of argon, the reaction system is continuously stirred for 18 hours and then stops reacting to obtain polystyrene nanoparticle emulsion;

13) in the reaction process, the liquid in the three-neck flask is gradually changed into thicker milky white liquid from thinner and bluish emulsion;

14) after the reaction is stopped, collecting the white emulsion of the product, transferring the white emulsion to a dialysis bag with the molecular weight cutoff of 10KD, dialyzing by taking ultrapure water as a dialysis medium, and replacing the dialysis medium for at least 3 times to remove the residual styrene and the sodium dodecyl sulfate;

15) after the dialysis, 1.00g of the liquid was removed and placed in an oven for constant weight, and the mass concentration of the polystyrene nanoparticle emulsion was determined.

2.2 copolymerization of styrene with 3- (trimethoxysilyl) propyl methacrylate

Through the copolymerization reaction of styrene and 3- (trimethoxysilyl) propyl methacrylate, a relatively hydrophilic polymer layer is covered on the surface of the hydrophobic polystyrene nano-particles. The experimental procedure was as follows:

1) diluting the milky white emulsion to a mass concentration of 10% by using ultrapure water;

2) transferring the diluted emulsion into a new 100mL three-necked flask, and respectively connecting a spherical condenser tube, an HD2010W constant-speed electric stirrer and a rubber plug to three bottle mouths;

3) inserting a needle head into a rubber plug of the three-neck flask, and introducing argon into the three-neck flask as in 4-5) in 2.1;

4) meanwhile, the mechanical stirring is maintained for 1h at a rotation speed of 400rpm by a HD2010W constant-speed electric stirrer;

5) taking down the needle head on the three-neck flask, and switching the guide pipe which is communicated with the argon gas to be communicated with the spherical condenser pipe to keep the ventilation;

6) uniformly mixing styrene and 3- (trimethoxysilyl) propyl methacrylate according to a volume ratio of 4:1, and fully swirling by using an MS2 type swirl mixer to obtain a mixed monomer solution, wherein the total mass of the mixed monomer solution is equivalent to the mass of the polystyrene nanoparticles in the emulsion;

7) adding the obtained mixed monomer solution into a reaction bottle through a 10mL injector, and continuously stirring for 1 h;

8) then, adding azodiisobutyronitrile which accounts for 3 percent of the mass of the mixed monomer solution as a reaction initiator, and continuously stirring and mixing for 1 hour;

9) opening an HS-70 type constant-temperature magnetic stirrer, heating to 70 ℃, and stopping the reaction after reacting for 18 hours to obtain MPS modified polystyrene nanoparticles;

10) after the reaction is finished, dialyzing and carrying out constant weight as in 13-14) in 2.1, and determining the mass concentration of the MPS modified polystyrene nanoparticle emulsion;

11) according to the measured mass concentration of the emulsion, it was diluted with ultrapure water to a mass concentration of 5% to prevent aggregation.

2.3 Synthesis of peanut-like nanoparticles

And further diluting the modified nano-particle emulsion to a mass concentration of 3.5%, and further swelling the nano-particle emulsion with a styrene monomer to obtain the peanut-shaped nano-particles. The method comprises the following specific steps:

1) weighing sodium p-styrenesulfonate which accounts for 0.8% of the mass of the nanoparticles, adding the sodium p-styrenesulfonate into a 100mL three-necked flask filled with the nanoparticle emulsion, and stirring for 15min to uniformly dissolve the sodium p-styrenesulfonate;

2) then a spherical condenser pipe, an HD2010W constant-speed electric stirrer and a rubber plug are respectively connected to the mouth of the flask;

4) meanwhile, the rotating speed of the HD2010W constant-speed electric stirrer is adjusted to 250rpm, and mechanical stirring is kept for 1 h;

5) uniformly mixing styrene monomer with volume being three times of that of the nano particles with azodiisobutyronitrile with mass concentration being 3%, and fully dissolving the mixture in a vortex mixer of MS2 type to obtain a swelling solution for later use;

6) adding the swelling liquid into a reaction system through a 10mL injector, and continuously stirring for 1h to achieve full swelling;

7) then, opening an HS-70 type constant-temperature magnetic stirrer, heating to 70 ℃, and stopping the reaction after reacting for 18 hours to obtain peanut-shaped nanoparticle emulsion;

8) after the reaction was completed, the precipitate was collected by ultracentrifuge (30000g, 30min) and redispersed in an equal volume of anhydrous ethanol by sonication in a water bath, and this process was repeated three times to remove residual unreacted monomer and initiator.

2.4 Synthesis of silica-modified peanut-like nanoparticles

By utilizing the different hydrophilicity of the two hemispheres of the peanut-shaped nanoparticles synthesized in the previous step, the silane hydrolysis condensation reaction only occurs on the hemispheres containing 3- (trimethoxysilyl) propyl methacrylate. The method mainly comprises the following steps:

1) measuring 4mL of nanoparticle ethanol dispersion obtained by the reaction, and placing the nanoparticle ethanol dispersion in a 100mL round-bottom flask;

2) adding 6mL of absolute ethyl alcohol into the round-bottom flask, further diluting to 10mL, and adding 0.5mL of 25% concentrated ammonia water;

3) adjusting an HS-70 type constant-temperature magnetic stirrer, and keeping stirring at the rotating speed of 500 rpm;

4) accurately weighing 0.30g of tetraethoxysilane, uniformly mixing the tetraethoxysilane with absolute ethyl alcohol with equal mass, and fully swirling by using an MS2 type vortex mixer for later use;

5) slowly injecting the mixed solution of tetraethoxysilane and absolute ethyl alcohol into the reaction solution by using a peristaltic pump at the flow rate of 1mL/h until the mixed solution is completely injected, and continuously stirring for 1 h;

2.5 Synthesis of the Nano bowl

And finally, removing the polymer template by utilizing the characteristic that the polystyrene is dissolved in tetrahydrofuran to obtain the nano-particles with the bowl-shaped structures. The specific experimental steps are as follows:

1) opening a water bath kettle of the R-200 rotary evaporator, and setting the water temperature to 45 ℃;

2) connecting the round-bottom flask filled with the nanoparticle ethanol dispersion liquid after the reaction is finished to a rotary evaporator, adjusting the rotating speed to a medium speed, and maintaining the rotary evaporation for 1h to ensure that the ethanol is completely volatilized;

3) after removing the ethanol, taking down the round-bottom flask, and adding a proper amount of tetrahydrofuran;

4) putting the round-bottom flask into a water bath for ultrasonic treatment for 5min, and uniformly dispersing the rotary-steamed dry powder;

5) placing the round-bottom flask on an HS-70 type constant-temperature magnetic stirrer, and keeping stirring at a high speed (1200rpm) for 24 hours;

6) the reaction solution was collected, the precipitate was collected by ultracentrifuge (30000g, 30min), and redispersed in triple distilled water by water bath sonication, which was repeated three times.

Secondly, calculating the mass concentration and the synthesis yield of the nano-particle emulsion obtained by the reactions in each step

Carrying out constant weight operation on the nanoparticle emulsion obtained by the reaction in each step:

1) selecting 3 clean EP empty tubes with the volume of 1.5mL, and recording the initial tube weight by the constant weight of the oven;

2) precisely weighing three parts of 1.00g of each nano-particle emulsion, adding the nano-particle emulsion into an EP tube with constant weight, and putting the EP tube into an oven;

3) taking out the EP tube at regular time, weighing, recording, and continuously putting back to the oven until the final two-time mass is unchanged, namely reaching the constant weight end point;

4) calculating the mass of the nano particles by a difference weight method, and calculating the mass concentration, wherein the calculation formula is as follows:

5) calculating the yield of each synthesis step according to the calculated mass concentration, wherein the calculation formula is as follows:

third, nano-particle characterization method

1. And (3) characterization of the nanoparticles by DLS and Zeta: after the nanoparticles were ultra-pure dispersed and diluted to the appropriate concentration, the light scattering particle size and Zeta potential were determined by a Malvern zetasizer nano ZS laser particle sizer.

2. Diluting the nanoparticle solution prepared in the key steps of the method, dropwise adding the diluted nanoparticle solution on the reinforced copper mesh, and observing the form of the nanoparticles by a CM-120 transmission electron microscope.

3. Nano bowl opening angle estimation

1) The ratio of the radii of the two hemispheres of the peanut-shaped nanoparticles (r) was measured by transmission electron microscopy₁:r₂) And the aspect ratio (L: W) of the nanoparticle as a whole, wherein W is 2 r₂。

2) According to the radius ratio and the length-width ratio, the opening angle of the nanometer bowl after the polystyrene is dissolved is calculated, and the calculation formula is as follows:

fourthly, the result

1. Nanoparticle emulsion mass concentration

The results of the concentration of each nanoparticle emulsion calculated according to the mass concentration calculation formula are shown in table 4, the synthesis yield is kept above 85%, and the synthesis efficiency is high.

TABLE 4 emulsion mass concentration and Synthesis yield of each nanoparticle

2. Particle size distribution of nanoparticles and Zeta potential

The particle size distribution and the Zeta potential of each nanoparticle are shown in FIG. 2: the polystyrene nanoparticles are successfully synthesized by an emulsion polymerization method, the size average value is 50.8nm, the PDI is 0.068, and the particle size uniformity of the nanoparticles is good, and the Zeta potential is-40.7 mV. Through the copolymerization reaction of styrene and 3- (trimethoxysilyl) propyl methacrylate, the average size of the finally obtained MPS modified polystyrene nanoparticles is 68.5nm, and the polystyrene nanoparticles are more electronegative due to the action of the 3- (trimethoxysilyl) propyl methacrylate, and the Zeta potential is-49.2 mV. And further stirring the modified nano particles and a styrene monomer, and performing swelling action and polymerization reaction to obtain peanut-shaped nano particles, wherein the average size value of the peanut-shaped nano particles is about 137.8nm, and the Zeta potential rises to a certain extent at-31.6 mV due to the increase of the styrene content. On the basis, slowly adding tetraethoxysilane dropwise by using a peristaltic pump, and further increasing the size of the nano-particles to 154.7nm after hydrolysis condensation reaction, wherein the Zeta potential is-39.5 mV. Finally, the polystyrene template was dissolved due to the addition of tetrahydrofuran, and the final product size was 126.7nm, which is significantly reduced from the previous silica-modified peanut-like nanoparticle size. Meanwhile, the Zeta potential is kept around-30.2 mV.

Specific values of the particle diameter and the Zeta potential obtained by the DLS detection of the nanoparticles in the steps are summarized in a table 5:

table 5 particle size and surface potential data for DLS measurements of each nanoparticle (n ═ 3)

3. Transmission electron micrograph of nanoparticles

The nanoparticles in the key reaction step are diluted to a proper concentration, the nanoparticle dispersion liquid is dripped on a carbon-supported film copper net, and after the nanoparticle dispersion liquid is naturally dried, a transmission electron microscope is used for observing a sample, and the results are shown in fig. 3 and 4.

As can be seen from FIG. 3.A, the synthesized polystyrene nanoparticles have good monodispersity and uniform size, the size is about 50nm, and the result is consistent with the DLS detection result. Because the synthesis process of the MPSNPs has no obvious change on the shape of the nano-particles and the size of the nano-particles has no obvious increase, the success of the synthesis of the MPSNPs cannot be accurately proved by means of a transmission electron microscope. Therefore, in this embodiment, the corresponding transmission electron microscope picture is not added, but the particle size and Zeta potential are detected by DLS and the subsequent transmission electron microscope image observation of the nanoparticles is performed to verify the synthesis result of the MPSNPs.

As the reaction proceeds, the swelling effect of styrene causes a significant change in the nanoparticle morphology based on MPSNPs, as shown in fig. 3.B, the nanoparticle is transformed from a regular round sphere to a peanut-like nanoparticle structure formed by connecting two hemispheres. Meanwhile, it can be found that, since the nanoparticles are transformed from spheres to rod-like irregularities, the particle size thereof should be measured by the longitudinal length thereof and the transverse width thereof at the same time. In combination with the detection result of the particle size in DLS, it is easy to find that the numerical value is closer to the longitudinal length of the nano-particles in the transmission electron microscope. And it can be inferred from transmission electron microscopy images that the lateral width of the nanoparticles is slightly increased compared to the first polystyrene nanoparticles. Successful synthesis of peanut-like nanoparticles and approximation of the width of the peanut-like nanoparticles to polystyrene nanoparticles were laterally validated for successful preparation of MPSNPs.

After introducing TEOS, the peanut-like nanoparticles were covered with silicon dioxide on the hemispheres containing MPS groups. Meanwhile, because tiny silicon dioxide nanoparticles are generated in the reaction environment, although the generation of the silicon dioxide nanoparticles can be effectively reduced by reducing the adding speed of TEOS through a peristaltic pump, the generation of the silicon dioxide nanoparticles cannot be completely avoided. When the tiny silica nanoparticles react with the peanut-like nanoparticles, the surface of the silica-modified peanut-like nanoparticles is not smooth, as shown in fig. 3.C, which is rougher than that shown in fig. 3.B, and tiny protrusions on the surface can be observed.

Finally, under the action of tetrahydrofuran, after the polystyrene is dissolved, one hemisphere in the original peanut-shaped nanoparticles can be clearly seen to disappear under a transmission electron microscope, and the nanoparticles are converted into a sphere. It is noted that the center of the remaining half sphere has a significantly lower electron density than the periphery, and appears pale gray than the periphery, indicating that the center of the sphere has also been hollowed out, leaving only a silica shell formed by the hydrolytic condensation of TEOS. Moreover, from the transmission electron microscope image, the nano bowls with different opening orientations can be clearly observed (fig. 4). The above evidences all prove the successful synthesis of the nanometer bowl.

4. Radius ratio, aspect ratio and nano bowl opening angle of peanut-shaped nanoparticles

1) Radius ratio and aspect ratio of peanut-shaped nanoparticles

By measuring the length and width of peanut-shaped nano particles in a transmission electron microscope and the radius length of two hemispheres, r is obtained after calculation₁:r₂≈9:10，L:W≈3:2。

2) Opening angle of nanometer bowl

According to the length and the width of the peanut-shaped nano particles obtained by the measurement and the radius length of the two hemispheres, the opening angle (theta) of the nano bowl is calculated to be in the range of 90-110 degrees by using an opening angle calculation formula.

Fifth, discuss

1. Selection and control of polystyrene nanoparticle size

As a seed template in preparation, subsequent reactions are carried out on the basis of the completion of the polystyrene nanoparticles, so that the preparation of the polystyrene nanoparticles directly influences the successful development of the subsequent reactions, and the synthesis of high-quality polystyrene nanoparticles is extremely important. Because the subsequent reaction needs to use a single polystyrene nanoparticle as a template, the requirement on the uniformity and monodispersity of the polystyrene nanoparticle is high. Any adhesion or agglomeration may affect the subsequent reactions, resulting in the final nanoparticles with altered properties, altered morphology and even failed synthesis. Meanwhile, the size selection of the polystyrene nanoparticles will also affect the overall size of the final nano bowl and the volume of the inner water cavity. According to an EPR effect theory, finally selecting and synthesizing the polystyrene nano particles within the range of 30-50nm as a seed template, and obtaining the final nano bowl after multi-step reaction, wherein the whole size of the nano bowl is about 100nm and the nano bowl has an inner water cavity with the diameter of 30-50 nm. According to the characteristics of emulsion polymerization, the volume of emulsion droplets in a reaction system can be changed by changing the feeding amount of an emulsifier SDS, so that the volume of styrene coated in the emulsion droplets is influenced, and the size of polystyrene nanoparticles is finally determined. Attempts have been made herein to select two different loadings of SDS (1g, 2g) to synthesize polystyrene nanoparticles of different sizes. As a result, when the dosage of SDS is increased, the size of the polystyrene nano-particles is remarkably reduced to about 30nm, the DLS detection result shows that 38.74 +/-12.7 nm and the Zeta potential is-40.9 +/-3.1 mV, while the PDI result shows that 0.372 +/-0.008. Meanwhile, the polystyrene nanoparticles observed by a transmission electron microscope show more obvious agglomeration and adhesion (fig. 5). Both the PDI and the tem results show that although increasing the amount of SDS can reduce the size of the polystyrene nanoparticles, the surface energy of the nanoparticles is significantly increased due to the reduction in size, resulting in significantly reduced stability of the nanoparticles and a tendency to agglomerate. Therefore, the polystyrene nanoparticles with the size of 50nm are finally selected as the seed templates of the subsequent reaction after consideration.

2. Swelling effect of styrene and synthesis principle of peanut-shaped nanoparticles

In the swelling process, the mixing and stirring time of styrene monomer and MPS modified polystyrene nanoparticles in advance is important. The polystyrene nanoparticles located in the center of the MPS-modified polystyrene nanoparticles can only be caused to volumetrically expand by allowing the styrene monomer to penetrate into the MPS-modified polystyrene nanoparticles with sufficient stirring time. And the volume expanded core polystyrene nanoparticle gradually cracks the coating shell of the MPS and styrene copolymer to form a gap. Subsequently, the temperature of the reaction system is increased, and under the initiation action of AIBN, the rest styrene monomer in the reaction system rapidly undergoes explosive polymerization reaction at the gap to form another hemisphere of the peanut-shaped nano particle at the gap. It is noted that, during the polymerization reaction, the styrene monomer cannot be sufficiently reacted with the MPS due to the hydrophobic property of the styrene and the hydrophilic property of the MPS, and the polystyrene at the gap has the same hydrophobic property as the styrene monomer, so that the styrene monomer is more apt to be rapidly polymerized at the gap according to the principle of similar compatibility.

3. Styrene swelling ratio and peanut-shaped nanoparticle hemisphere size control

By adjusting the addition of the styrene monomer, the size of one hemisphere generated by swelling of one of the peanut-shaped nanoparticles can be regulated. The above demonstration is verified by selecting two different styrene monomers and MPS modified polystyrene nanoparticles in a ratio of 3:1 and 9:1, respectively. The results are shown in the transmission electron microscope of FIG. 6, when the charging volume ratio (V) of styrene monomer to MPS modified polystyrene nano-particles_styrene:V_MPSNPs) At 3:1, the synthesized peanut-like nanoparticles were comparable in size of both hemispheres (fig. 6. a). After the Vstyrene VMPSNPs is increased to 9:1, the size of two hemispheres of the synthesized peanut-shaped nanoparticles is far from each other (FIG. 6.B), wherein the smaller hemisphere is the original MPS modified polystyrene nanoparticle, and the size of the other hemisphere is obviously increased due to the increase of the styrene monomer, and the diameter of the other hemisphere is already increasedUp to 200nm, which is 4 times the size of the smaller hemisphere. Whereas subsequent reactions require deduction of polystyrene, excessive polystyrene proportions may result in incomplete final dissolution processes, thereby affecting the synthesis of the final nanobowl. Therefore, V is selected_styrene:V_MPSNPsThe feeding ratio of 3:1 is taken as a scheme for finally synthesizing the peanut-shaped nano particles.

4. Selective hydrolytic condensation of TEOS and control of flow rate

After successful synthesis of peanut-like nanoparticles, TEOS was added for hydrolytic condensation reaction. Since the synthesized peanut-like nanoparticles are not only specially shaped, but also have completely different surface groups of two hemispheres. Due to the addition of MPS, one hemisphere of the peanut-shaped nanoparticle contains silanol groups, and the other hemisphere formed later is polymerized by a simple styrene monomer, and the surface of the hemisphere has only an olefinic bond of styrene, but does not contain silanol groups. Thus, under the alkaline conditions of aqueous ammonia, TEOS passes through

And reacting to perform hydrolytic condensation reaction with silanol group on MPS. And the other hemisphere cannot generate hydrolytic condensation reaction because of no silanol group, so that the selective modification of the silicon dioxide and the hemisphere containing MPS is realized. It should be noted that TEOS itself can be hydrolyzed and condensed under the catalytic action of ammonia water to form silica nanoparticles. The correct TEOS addition rate is a very critical influencing factor in order to reduce the probability of TEOS self hydrolytic condensation. By reducing the adding speed of TEOS as much as possible, the added TEOS reacts with peanut-shaped nano particles rapidly, thereby reducing the silicon dioxide nano particles formed by self condensation. However, although this operation can reduce the probability of silica nano-generation, it is still unavoidable to form minute silica nanoparticles and continue condensation with peanut-like nanoparticles in the form of minute silica nanoparticles. Thus, there is a phenomenon as described above, and the surface of the silica-modified peanut nanoparticles is not smooth.

5. Feeding amount of TEOS and shape of nano bowl

Finally, the present embodiment also considers the relationship between the feeding amount of TEOS and the shape of the nanometer bowl. And respectively selecting 0.3g and 0.5g of TEOS (tetraethyl orthosilicate) inventory to prepare nano particles, respectively dripping the nano particles on a copper mesh after obtaining the final nano bowl, and observing the shape difference of the nano bowl through a transmission electron microscope. As shown in fig. 7.a, when the amount of TEOS added was 0.3g, the silica layer on the surface of the nanoparticles could be clearly observed, and the structures of the bowl mouth and the bowl wall could be clearly distinguished. However, when the amount of TEOS added is increased to 0.5g, the probability of hydrolysis and condensation of TEOS itself is significantly increased due to the increase of TEOS, and the generation of silica nanoparticles is significantly increased while forming a nano bowl, as shown in fig. 7.B, which is a large increase in the ratio of solid silica nanoparticles compared to fig. 7. a. Therefore, to obtain more nanobowls, 0.3g of TEOS was chosen as the final solution for preparing silica-modified peanut-like nanoparticles.

Six and small knot

The construction method of the nanometer bowl comprises the following steps: taking styrene monomer as a raw material, and synthesizing polystyrene nano-particles in an emulsion polymerization manner; then adding MPS/styrene mixed solution mixed according to a certain proportion, and preparing MPS modified polystyrene nano-particles through copolymerization reaction; on the basis, adding a styrene monomer again, and obtaining peanut-shaped nano particles by utilizing the swelling characteristic and polymerization reaction of styrene; transferring the prepared peanut-shaped nano particles into absolute ethyl alcohol for uniform dispersion, adding TEOS, and selectively reacting with a hemisphere containing MPS groups in the alkaline environment of concentrated ammonia water

Reacting to form silica-modified peanut-shaped nanoparticles; finally, the polystyrene component in the nanoparticles is dissolved by utilizing the characteristic that the polystyrene is dissolved in tetrahydrofuran, and only the shell containing silicon dioxide is left, so that the nanoparticles with a bowl-shaped structure are obtained. The charge ratio of each reaction step is determined by optimizing conditions, and finally the nanometer bowl with the average grain diameter of 126.7nm and the Zeta electricity is preparedThe nano-particles are positioned at-30.2 mV, the PDI is 0.142, the size of the nano-particles is uniform, and the monodispersity is good. The morphological structure of the nanometer bowl is verified through observation of a transmission electron microscope; and the opening angle of the nanometer bowl is estimated to be in the range of 90-110 degrees through the radius ratio and the length-width ratio of the nanometer particles.

Example 2 preparation of nano bowl-supported liposome and establishment of drug loading method

In this example, the liposome and the nano bowl prepared in example 1 were combined by electrostatic adsorption by means of probe ultrasound. And then, carrying out active drug loading by adopting an ammonium sulfate gradient method to finish the high-entrapment-rate loading of the alkalescent drug adriamycin to obtain the nano bowl-supported adriamycin liposome.

The specific synthetic route is schematically shown in FIG. 8.

Firstly, preparing the nano bowl supporting liposome

1 instruments and materials

1.1 instrumentation

TABLE 6

1.2 materials and reagents

TABLE 7

2 method of experiment

2.1 amination modification of Nano bowl

1) Taking 20mg of the nano bowl ultrapure water dispersion liquid prepared in the previous step, and transferring and dispersing the nano bowl ultrapure water dispersion liquid into 10mL of absolute ethyl alcohol through ultracentrifugation (30000g for 30 min);

2) adding the absolute ethyl alcohol dispersion liquid containing the nano bowl into a 25mL round-bottom flask, and stirring at a constant speed;

3) adding 200 μ L of APTES into the dispersion, and stirring at 150rpm overnight;

4) after the reaction is finished, taking out the reaction solution, removing unreacted APTES by centrifugation (24000g, 30min), collecting the precipitate, and dispersing in 5mL of ultrapure water, wherein the process is repeated for 3 times;

5) DLS detects the particle size and the potential of the nanoparticles.

2.2 preparation of Nano bowl-supported liposomes

1) Accurately weighing 25mg of cholesterol, HSPC and DSPE-PEG2000 respectively, and dissolving in 1mL of chloroform respectively to obtain 25mg/mL of lipid solution;

2) fully dissolving 10mg of DiR in 1mL of trichloromethane;

3) taking a proper amount of the solution according to the mass ratio of HSPC: Chol: DSPE-PEG2000 to DiR: 3:1:1 (when preparing the liposome containing DiR, the mass ratio of HSPC: Chol: DSPE-PEG2000 to DiR: 3:1:1: 0.05) to ensure that the total mass of the lipid is 20mg, adding a proper amount of trichloromethane, diluting to 5mL, and uniformly mixing the solution by an MS2 type vortex mixer;

4) preparing liposome by adopting a film dispersion method: adding the uniformly mixed lipid solution into a 500mL round-bottom flask, connecting the flask to a rotary evaporator, adjusting the medium rotation speed, keeping the water temperature at 40 ℃, performing rotary evaporation for 1h, removing trichloromethane, and uniformly covering the lipid on the bottom of the round-bottom flask;

5) taking down the round-bottom flask, putting the round-bottom flask into a vacuum drying oven, and drying the round-bottom flask overnight;

6) preparation of 300mM, pH 5.5 ammonium sulfate solution: precisely weighing 7.93g of ammonium sulfate, and fully dissolving the ammonium sulfate in 200mL of ultrapure water; adjusting the pH value to 5.5 by 1M sodium hydroxide solution;

7) transferring the aminated nano bowl synthesized in the step 2.1 into a prepared ammonium sulfate solution by a centrifugal means (24000g, 30 min);

8) adding ammonium sulfate solution containing aminated nanometer bowl into round-bottom flask after drying overnight, placing into ZQTY-70 shaking incubator, and shaking (250rpm, 60 deg.C) for 1 hr to fully hydrate lipid to form vesicle coated with nanometer bowl;

9) fixing the obtained liposome vesicle suspension on a JY 92-II ultrasonic cell crusher, and carrying out probe ultrasonic for 30min in a mode of working for 15 seconds and pausing for 15 seconds at 35W power to prepare the uniformly dispersed nano bowl supported liposome.

2.3 preparation of Normal liposomes

1) Thin film dispersion of lipids was prepared with reference to 1) -6) of 2.2;

2) transferring 5mL of prepared ammonium sulfate solution, adding into the round-bottom flask after drying overnight, placing into a ZQTY-70 shaking incubator, and shaking (250rpm, 60 ℃) for 1h to fully hydrate the lipid to form vesicle suspension;

3) assembling a liposome mini extrusion device, installing a PC membrane with the aperture of 200nm, and extruding the obtained vesicle suspension back and forth for 20 times through the extrusion device to improve the uniformity of the liposome size.

2.4 entrapment of doxorubicin hydrochloride

1) A 10% sucrose dialysate containing 10mM histidine at pH 6.5 was prepared: weighing 500g of sucrose, dissolving in 5L of ultrapure water, adding 7.75g of histidine, and fully dissolving; adjusting the pH value to 6.5 by 1M hydrochloric acid solution;

2) cutting two sections of 3.5KD dialysis membranes, respectively adding the liposome supported by the nanometer bowl and the common liposome prepared in the previous step, putting the two into a 1L beaker containing sucrose dialysate, and dialyzing, wherein the solution is changed for 4 times;

3) precisely weighing 2.0mg of doxorubicin hydrochloride, and dissolving the doxorubicin hydrochloride in 1mL of sucrose dialysate;

4) respectively adding 0.5mL of doxorubicin hydrochloride/mL sucrose solution into the two groups of liposomes after dialysis, and performing air-blow beating for several times to fully and uniformly mix;

5) putting the mixed solution of the two groups of liposomes and the doxorubicin hydrochloride into a ZQTY-70 shaking incubator for shaking (250rpm, 60 ℃) for 1 h;

6) placing the liposome supported by the nanometer bowl into a centrifuge, collecting precipitate by centrifugation (24000g, 30min), redispersing with 10% sucrose dialysate, and washing for three times to remove free doxorubicin hydrochloride and liposome not encapsulated in the nanometer bowl; the normal doxorubicin liposomes were dropped onto the G-50 sephadex, and the free doxorubicin hydrochloride was eluted.

Secondly, establishment of doxorubicin hydrochloride fluorescence quantitative analysis method

1. Selection of detection wavelength

1) Weighing 1mg of doxorubicin hydrochloride, and dissolving the doxorubicin hydrochloride in 10mL of ultrapure water;

2) transferring 200 μ L of sample, and dropping into 96-well plate;

3) performing ultraviolet absorption scanning within the range of 200-850nm by using a SpectraMax M2 biomolecular enzyme labeling instrument to obtain an ultraviolet absorption spectrum of the doxorubicin hydrochloride;

4) and determining the maximum absorption wavelength Dox-max of the doxorubicin hydrochloride by using the ultraviolet absorption spectrum, detecting the fluorescence emission spectrum of the doxorubicin hydrochloride by using the wavelength as the excitation light wavelength, and obtaining the maximum fluorescence emission wavelength Em Dox-max of the doxorubicin hydrochloride.

2. Drawing of standard curve

1) Precisely weighing a proper amount of adriamycin hydrochloride, preparing stock solution with a certain concentration by using ultrapure water, and then continuously diluting the stock solution by using the ultrapure water to obtain adriamycin hydrochloride reference substance solutions of 0.156, 0.313, 0.625, 1.250, 2.500, 5.000 and 10.000 mu g/mL;

2) according to the Dox-max and Em Dox-max obtained by the method, a fluorescence quantitative analysis detection method is established: detecting the fluorescence emission intensity of the doxorubicin hydrochloride reference substance solution with each concentration at the wavelength of Em Dox-max by taking Dox-max as the excitation light wavelength;

3) and performing linear regression on the concentration of the doxorubicin hydrochloride by using the fluorescence emission intensity to obtain a fluorescence quantitative standard curve equation of the doxorubicin hydrochloride.

3. Precision degree

1) Selecting the doxorubicin hydrochloride reference substance solutions of 0.156, 1.250 and 10.000 mu g/mL as samples with low concentration, medium concentration and high concentration respectively;

2) detecting the fluorescence intensity of doxorubicin hydrochloride at each concentration for 2, 4 and 8 hours respectively to calculate standard deviation within day (RSDIntra-day); the detection was performed at 1d, 2d, and 3d, respectively, and the standard deviation between days (RSD Inter-day) was calculated.

4. Recovery rate

1) Selecting 3 parts of the doxorubicin hydrochloride reference substance solutions of 0.156, 1.250 and 10.000 mu g/mL respectively, and detecting the fluorescence intensity;

2) substituting the obtained fluorescence intensity into a standard curve formula, and calculating to obtain the doxorubicin hydrochloride determination concentration;

3) the recovery rate was calculated from the measured concentration and the actual concentration.

Three, double-fluorescence qualitative detection of nano particles

1. Selection of DiR detection wavelength

1) Transferring 20 mu L of 10mg/mL DiR chloroform solution, and diluting to 1mL by using methanol;

2) transferring 200 μ L of sample, and dropping into 96-well plate;

3) performing ultraviolet absorption scanning within the range of 200-850nm by using a SpectraMax M2 biomolecular enzyme labeling instrument to obtain an ultraviolet absorption spectrum of the DiR;

4) and determining the maximum absorption wavelength DiR-max of the DiR through the ultraviolet absorption spectrum, selecting a wave band with the minimum influence on the fluorescence spectrum in the DiR ultraviolet absorption region as the excitation light wavelength, detecting the fluorescence emission spectrum of the DiR, and obtaining the maximum fluorescence emission wavelength Em DiR-max of the DiR.

2. Nanoparticle ultraviolet absorption spectrum and dual-fluorescence signal emission spectrum

1) Respectively transferring a proper amount of nano bowl support liposome or common adriamycin liposome which is loaded with adriamycin and is embedded with DiR in a liposome phospholipid bilayer, and adding the nano bowl support liposome or the common adriamycin liposome into a corresponding 96-well plate;

2) performing ultraviolet absorption scanning within the range of 200-850nm by using a SpectraMax M2 biomolecular enzyme-labeling instrument to obtain an ultraviolet absorption spectrum of the nanoparticles;

3) and then, according to the method, respectively selecting the maximum absorption wavelength Dox-max of the doxorubicin hydrochloride and the wave band with the minimum influence on the fluorescence spectrum in the ultraviolet absorption region of the DiR as the excitation light wavelength, and detecting the fluorescence emission spectra of the doxorubicin hydrochloride and the DiR in the nanoparticles.

Fourthly, determining the encapsulation efficiency and drug loading capacity of the liposome

1) Precisely transferring 50 mu L of nano bowl supporting liposome or common adriamycin liposome sample loaded with adriamycin, and diluting to 1mL by ultrapure water;

2) adding a 0.2% Triton solution to the sample to allow lysis of the liposomes, releasing doxorubicin in the inner aqueous cavity;

3) detecting the fluorescence intensity of the adriamycin by a fluorescence quantitative analysis method;

4) substituting the fluorescence intensity into a standard curve equation to obtain the adriamycin concentration, and calculating the encapsulation efficiency by the following specific formula:

5) precisely transferring 1mL of a nano bowl-supported adriamycin liposome or a common adriamycin liposome sample, filling the sample into an EP tube subjected to constant weight treatment, putting the EP tube into a refrigerator at the temperature of-80 ℃ for overnight freezing, and transferring the sample into an RLPHR 2-4LD freeze dryer for freeze drying.

6) Weighing the freeze-dried sample, and calculating the drug loading:

five results

1. Amination modification characterization of nanometer bowl

After amination, the DLS result shows that the size of the nanometer bowl is not changed significantly, but the Zeta potential of the nanometer bowl is turned from the original-30.2 +/-1.1 mV to +34.5 +/-1.5 mV, as shown in FIG. 9. C. The reversal of the potential proves that the surface of the nanometer bowl is successfully modified with amino, so that the Zeta potential of the nanometer bowl is positive. Meanwhile, the transmission electron microscope result of fig. 9.a shows that the morphology of the nanoparticles is not changed, and the bowl-shaped structure is not affected.

2. Characterization of Nanowbowl-supported liposomes and common liposomes

By adding the liposome, DLS detection shows that the size of the nano bowl supporting liposome is increased to 143.6 +/-6.2 nm, and is increased by nearly 20nm compared with the size of the nano bowl. Meanwhile, the Zeta potential result showed-17.9. + -. 0.3 mV. The common liposome extruded by a 200nm PC film has the particle size of 148.3 +/-2.9 nm and the Zeta potential of-20.3 +/-1.2 mV. The nano bowl supports the slight increase of liposome particle size and the re-reversal of Zeta potential and approaches the common liposome. Subsequently, a nano particle sample negatively dyed by uranyl acetate is observed under a transmission electron microscope, a circle of phospholipid bilayers around the nano bowl can be clearly seen, and meanwhile, the nano bowl wrapped in the liposome is still clearly visible. The successful encapsulation of liposomes was verified by the above phenomena.

3. Ultraviolet absorption spectrum and fluorescence spectrum

3.1 ultraviolet absorption and fluorescence spectra of doxorubicin hydrochloride and DiR

The ultraviolet absorption spectra of doxorubicin hydrochloride and DiR were plotted from the results of wavelength scanning with a microplate reader, and as shown in fig. 11, Dox-max was 480nm and DiR-max was 740 nm. Then 480nm and 700nm are respectively taken as the excitation light wavelength of the two, and a corresponding fluorescence emission spectrum is obtained by using a microplate reader, wherein Em Dox-max is 580nm, and Em DiR-max is 780 nm.

3.2. Nanoparticle ultraviolet absorption spectrum and dual fluorescence emission spectrum

The ultraviolet absorption and fluorescence signals of the two prepared liposomes are measured, the result is shown in figure 12, no matter the ultraviolet absorption spectrum or the fluorescence emission spectrum, the characteristic absorption peak and emission peak of doxorubicin hydrochloride and DiR can be found, the coexistence of the liposome and the doxorubicin in the nanoparticles in the system is demonstrated, and the successful entrapment of the doxorubicin is proved. By carefully comparing the ultraviolet absorption spectra of the NB @ DLP group and the DLP, the NB @ DLP can be found to have significantly higher absorption at 300-400nm than the DLP, and the waveband range is presumed to be the non-characteristic absorption region of the nanometer bowl.

4. Nanoparticle encapsulation efficiency and drug loading measurement

4.1. Fluorescent quantitative standard curve of doxorubicin hydrochloride

According to the detection result of the microplate reader, the fluorescence intensity value of doxorubicin hydrochloride at 580nm wavelength of each concentration is recorded, and the fluorescence intensity RFU is used for carrying out linear regression on the concentration C (mu g/mL) to obtain the RFUDox which is 99.11C-9.871, R²0.998. At the same time, examine the methodThe results of the precision and recovery of (D) are shown in tables 8 and 9. The precision RSD in the day and the day is less than 3 percent, which shows that the method has good precision.

TABLE 8 precision test

TABLE 9 recovery test

4.2. Nanoparticle encapsulation efficiency and drug loading measurement

The fluorescence intensity of doxorubicin hydrochloride in the nanoparticles is obtained by measuring with a microplate reader, the fluorescence intensity is substituted into a doxorubicin hydrochloride fluorescence quantitative analysis standard curve to obtain the concentration of doxorubicin hydrochloride in the corresponding nanoparticles, and the drug loading rate and encapsulation rate results are calculated and shown in table 10. The results show that the existence of the nanometer bowl support has no significant influence on the drug loading efficiency of the liposome, and the entrapment rate is kept at about 90%.

Table 10 nanoparticle encapsulation efficiency and drug loading (n ═ 3)

Sixth, discuss

1. Influence of nanometer bowl Zeta potential and particle size on liposome fusion entrapment

The fusion and entrapment process of the nano particles and the liposome is a result caused by various factors. Research shows that the Zeta potential and the size of the nano-particles influence the final fusion result. In the fusion coating process of the nano particles and the liposome, the electrostatic adsorption force plays a crucial role. When the Zeta potential of the liposome is negative, if the Zeta potential of the nano particles is opposite to positive, the liposome and the nano particles can be quickly approached to each other under the action of electrostatic adsorption force, and are adsorbed, and finally, the nano particles are fused with the liposome and enter the water cavity in the liposome; when the Zeta potential of the nanoparticle is also negative, due to the same electrical property between the nanoparticle and the liposome, the rapidly increased electrostatic repulsion force when the nanoparticle and the liposome approach each other can cause the nanoparticle and the liposome to be unable to approach each other, thereby hindering the fusion of the nanoparticle and the liposome. At the same time, the size of the nanoparticles will also affect the fusion with the liposomes. When the size of the nanoparticle is far smaller than that of the liposome, although the opposite electrical property can promote the approach and adsorption of the nanoparticle and the liposome, because the size of the nanoparticle is too small, the surface potential of the nanoparticle is not enough to enable the nanoparticle and the liposome to be fused, and on the contrary, a large number of small nanoparticles exist on the surface of the liposome, and meanwhile, the small nanoparticles can continuously adsorb other liposomes, so that cross-linking is formed to destroy the dispersion balance of the nanoparticle, and finally, sedimentation is caused; when the size of the nanoparticles is comparable to that of the liposome, the surface potential will be sufficient to initiate the fusion of the nanoparticles with the liposome and form a stable association.

2. Selection of model drug doxorubicin hydrochloride

The doxorubicin hydrochloride is used as an anti-tumor treatment drug, belongs to an anthracycline broad-spectrum anti-tumor drug, and has strong cytotoxicity. Although doxorubicin hydrochloride is widely used in various types of antitumor therapy, it also brings many adverse reactions due to its strong cytotoxicity. Thus, for many years, pharmaceutical workers have been working on finding effective means of reducing the toxicity of doxorubicin hydrochloride, and preparing it into a corresponding formulation is a dosing regimen. In addition, due to the unique anthracene ring structure of the adriamycin, the adriamycin has strong red fluorescence characteristic, is beneficial to detection, and is convenient for the design and development of various experiments.

3. Selection of doxorubicin hydrochloride drug loading mode

When the nano bowl is designed to support the adriamycin liposome originally, a passive drug loading mode is hoped to be adopted, and a hydration liquid dissolved with a certain concentration of adriamycin hydrochloride is directly added in the liposome hydration process. However, through experiments, the encapsulation efficiency and the loading efficiency of the adriamycin are very low by the method. The reason for this is mainly that the volume of the inner water cavity of the liposome is far smaller than that of the outer water phase, so that most of doxorubicin hydrochloride is retainedIn the external aqueous phase and cannot be entrapped by the liposomes. Therefore, the passive loading method not only can not realize high-concentration drug loading, but also causes the waste of a large amount of free drugs. For this purpose, the active loading of doxorubicin hydrochloride is achieved here using a drug loading method similar to the FDA approved marketed Doxil prescription, using an ammonium sulfate gradient. As doxorubicin hydrochloride is a weakly basic drug, higher encapsulation efficiency can be realized by utilizing the weak acidity of ammonium sulfate. Firstly, hydrating by using a hydration solution containing ammonium sulfate to ensure that the water cavity in the liposome presents a weak acid environment; then, establishing a pH gradient between an outer aqueous phase and an inner aqueous cavity through dialysis; adding doxorubicin hydrochloride into the external aqueous phase, and generating NH by ionization equilibrium of ammonium sulfate₃Small molecules easily pass through a phospholipid bilayer and are neutralized with doxorubicin hydrochloride in an external water phase, so that doxorubicin is molecularly converted into a transmembrane material and enters a water cavity in a liposome; after entering the inner aqueous cavity of the liposome, doxorubicin reacts with H in the inner aqueous cavity⁺Combining and ionizing again to form salt; the formed adriamycin sulfate forms insoluble crystals in the inner water cavity, so that the transmembrane leakage of the adriamycin is prevented, and higher entrapment rate is realized. The detailed principle is shown in fig. 13.

Seven, small knot

The nano bowl supported liposome is prepared by incubating the nano bowl and the liposome and carrying the adriamycin successfully by using an active ammonium sulfate drug carrying method. Constructing a nanoparticle characterization evaluation system: DLS detection size and Zeta potential change, transmission electron microscope negative staining observation of phospholipid bilayer structure, enzyme labeling instrument determination of adriamycin and DiR double-labeled nanoparticle fluorescence signal, and encapsulation efficiency and drug loading determination. The average particle diameter of the final nano-particles is 143.6nm, and the Zeta potential is-17.9 mV. The transmission electron microscope results indicate that the nanoparticles are complete in shape and clear in structure, and can be successfully fused and coated with the liposome, so that the phospholipid bilayer of the liposome can be clearly observed. The ultraviolet absorption and fluorescence signals of the adriamycin and the DiR are detected by the nano particles in a microplate reader, and the successful construction of the nano particles is further proved. The encapsulation efficiency and drug loading were 89.57% and 2.34%, respectively. By means of active drug loading, the encapsulation efficiency and the drug loading rate of the adriamycin are obviously improved, and feasibility and theoretical basis are provided for clinical conversion.

Example 3 stability study and evaluation of in vitro antitumor therapy of Nanowan-supported Doxorubicin liposomes

Instrument and material

1. Instrumentation and equipment

TABLE 11

2. Materials and reagents

TABLE 12

3. Cell line and animal

4T1 Breast cancer cells, purchased from Caliper Life Sciences, Hopkinton, Mass., and used in this experiment at 3-5 passages.

4. Preparation of related reagents

1)4T1 cell culture fluid: DMEM basal medium, 1 streptomycin double antibody, 10% fetal bovine serum;

2) 4% paraformaldehyde solution: using PBS (0.01M) with pH 7.4 as a solvent, weighing 40g of paraformaldehyde, putting into 800mL of PBS solution, shaking and dissolving in a constant-temperature shaking table at 60 ℃ overnight, stopping heating after the paraformaldehyde solid is completely dissolved, balancing to room temperature, and adding PBS again to fix the volume to 1L.

Second, Experimental methods

1. Evaluation of stability of nano bowl-supported adriamycin liposome

1.1 evaluation of serum stability

1) Transferring appropriate amount of nanometer bowl supporting liposome and common adriamycin liposome loaded with adriamycin, respectively adding into 100% FBS, blowing uniformly, and placing into a constant temperature shaking incubator at 37 deg.C for uniform shaking at 120 rpm;

2) sucking the mixed solution in equal amount for 0, 2, 4, 6, 8, 12 and 24 hours respectively, and measuring the fluorescence intensity Ft of the adriamycin in the mixed solution by a microplate reader;

3) after 24h, adding 0.2% Triton solution, cracking and destroying liposome, completely releasing adriamycin, and recording fluorescence intensity F of adriamycin by using an enzyme-labeling instrument_final；

4) The leakage rate of the adriamycin in the whole blood serum is calculated according to the fluorescence intensity, and the detailed formula is as follows:

5) the nano bowl-supported doxorubicin liposomes at each time point were collected by centrifugation (24000g, 15min), and after redispersion, particle size and potential were determined by DLS.

1.2. Evaluation of Freeze drying stability

1) Precisely transferring an equivalent amount of a nano bowl-supported adriamycin liposome or a common adriamycin liposome sample, filling the nano bowl-supported adriamycin liposome or the common adriamycin liposome sample into an ep tube, putting the nano bowl-supported adriamycin liposome or the common adriamycin liposome sample into a refrigerator at minus 80 ℃ for overnight freezing, and transferring the nano bowl-supported adriamycin liposome or the common adriamycin liposome sample into an RLPHR 2-;

2) taking the freeze-dried nano bowl to support the adriamycin liposome or the common adriamycin liposome, adding the same amount of ultrapure water again, and performing vortex to fully disperse the adriamycin liposome or the common adriamycin liposome;

3) observing and recording the dispersion condition of the nano particles;

4) DLS measures the change of the particle size of the nanoparticles after re-dispersion after freeze-drying.

1.3 evaluation of storage stability

1) Placing the prepared nano bowl-supported adriamycin liposome dispersion liquid in a refrigerator at 4 ℃ for refrigeration;

2) DLS measurement of the particle size and Zeta potential was carried out on the corresponding dates, and changes in the long-term storage state of the nanoparticles were recorded.

2. 4T1 cell culture

2.14T1 cell recovery

1) Taking out the frozen 4T1 cells from the liquid nitrogen tank, incubating in a water bath at 37 ℃, and thawing;

2) after the solid in the freezing and storing tube is melted into liquid, transferring the liquid into a centrifuge tube, centrifuging for 5min at 800g, and removing supernatant freezing and storing liquid;

3) adding 5mL of cell culture solution containing 10% fetal calf serum, blowing the cells by a pipette to uniformly disperse the cells, and then filling the cells into a T-25 culture bottle;

4) the flask was placed at 37 ℃ and 5% CO₂The cells were cultured in a constant temperature incubator, and the growth state of the cells was observed under a microscope the next day.

2.2. Cell culture and passage

1) Removing the cell culture solution in the original T-25 culture flask by using a pipette, adding 1mL of DPBS (double stranded sequencing batch) for rinsing for 1-2 times to remove the residual culture solution;

2) adding 1mL of 0.25% trypsin, shaking the flask back and forth to allow the trypsin to cover the entire bottom surface of the flask, placing at 37 deg.C and containing 5% CO₂Incubating for 3min in the constant temperature incubator;

3) adding 4mL of cell culture medium containing 10% fetal calf serum, diluting pancreatin, and stopping digestion;

4) blowing the cell suspension by using a pipettor to uniformly disperse the cell suspension to obtain a single cell suspension, and mixing the cell suspension according to the weight ratio of 1: 4, subpackaging the mixture into a new T-25 culture bottle, and supplementing a fresh cell culture solution to 5 mL;

5) the flask was returned to 37 ℃ with 5% CO₂Culturing in a constant-temperature incubator, and observing the growth state of the cells under a microscope on the next day;

6) after 2 days, the liquid change passage operation was performed again.

2.3 cell cryopreservation

1) Removing the culture solution in the original T-25 culture flask by suction, adding 1mL of DPBS (double stranded sequencing batch) for rinsing for 1-2 times to remove the residual culture solution;

2) adding 500 μ L of 0.25% trypsin, shaking the flask back and forth to allow the trypsin to cover the entire bottom surface of the flask, placing at 37 deg.C and 5% CO₂Incubating for 3min in the constant temperature incubator;

4) blowing the cell suspension by using a pipettor to uniformly disperse the cell suspension to obtain a single cell suspension;

5) collecting the cell suspension in a sterile centrifuge tube, centrifuging at 800rpm for 5min, and removing the supernatant;

6) adding 1mL of cell cryopreservation solution, and blowing and beating the cell cryopreservation solution into single cell suspension;

7) transferring the single cell suspension to a freezing tube, marking, preserving at-80 ℃ overnight, and then transferring to a liquid nitrogen tank for preservation.

2.44T 1 Breast cancer cell uptake assay for Nanowu-supported Doxorubicin liposomes

1) The nano bowl-supported adriamycin liposome and the common adriamycin liposome are prepared by the method and the process;

2) after digestion of 4T1 cells, the cells were digested at 2X 10⁵The cell concentration of each/mL is inoculated in a small dish with a cover glass, the inoculation volume is 1mL, and each nanoparticle group is inoculated in 3 small dishes;

3) the cell-seeded dishes were placed at 37 ℃ in 5% CO₂The culture is carried out for 12 hours in a constant temperature incubator;

4) adding corresponding liposome dispersion solution with adriamycin concentration of 0.5mg/mL into each small dish, at 37 deg.C and 5% CO₂Continuously incubating for 1h in the constant-temperature incubator;

5) after 1h, the cell culture medium is aspirated, rinsed 3 times with DPBS, and then fixed for 20min by adding 1mL of 4% paraformaldehyde;

6) after fixation is finished, sucking out paraformaldehyde, rinsing with DPBS for 3 times, finally adding 1mL of DPBS solution containing 6 muL of DAPI, incubating for 5min, and rinsing with DPBS for 3 times;

7) observing the fixed sample under a laser confocal microscope, and selecting Alexa

The 488 channel observes the nanoparticle uptake by cells.

3. CCK-8 method for detecting cell viability

1) Taking 4T1 cells in logarithmic growth phase, counting, diluting to 2X 10 with complete cell culture medium⁵Density per mL and seeded into 96-well plates at 200 μ L per well. The plates were incubated at 37 deg.C,5％CO₂culturing in a constant-temperature cell culture box for 12 h;

2) preparing different nano-particle structures in a serum-free culture solution according to a drug concentration gradient to prepare a culture medium containing liquid drugs with different concentrations, sucking out old culture solution in a culture plate, and adding 200 mu L of newly prepared culture solution containing liquid drugs into each hole;

3) the cell-containing 96-well plate was again placed at 37 ℃ in 5% CO₂Culturing for a proper time in a constant-temperature cell culture box;

4) diluting CCK-8 liquid by 10 times with serum-free culture solution according to a ratio of 1:10, sucking out old culture solution in a culture plate, and adding 100 mu L of CCK-8-containing culture solution into each well;

5) the plates were incubated at 37 ℃ in 5% CO₂Incubating for 0.5-1h in a constant-temperature cell incubator;

6) the absorbance of each well was measured at a wavelength of 450nm using a microplate reader.

4. Statistical treatment

The results of the experiment are presented as "mean ± SD", statistically analyzed and graphed using GraphPad prism7.0 medical mapping software. The comparison between the two groups adopts t test; three or more groups of comparisons are statistically analyzed by a multi-factor analysis of variance method, and differences are considered to have statistical significance when p is less than 0.05.

Three, result in

1. Nanoparticle serum stability

The study was performed by dispersing nanoparticles in 100% fetal bovine serum and shaking-culturing in a 37 ℃ constant temperature shaking incubator to approximate the environment and blood flow impact of the nanoparticles in the circulatory system. The leakage rate of adriamycin is calculated by measuring the fluorescence intensity of adriamycin in different time. The result is shown in fig. 14.a, the leakage rate of the doxorubicin liposome supported by the nanometer bowl in the serum is significantly reduced, the average value of the leakage rate in 24h is 3.34%, and the leakage rate is kept below 5%, and almost no leakage is observed. However, the average leakage rate of the common adriamycin liposome is 23.00 percent, and the difference between the two is about 7 times. The result indicates that the leakage of the water cavity medicament in the liposome can be effectively reduced through the supporting effect of the nanometer bowl. Meanwhile, the change of the particle size and the Zeta potential of the liposome supported by the nanometer bowl in serum within 24h is examined, and the result is shown in fig. 14.B, and no obvious change occurs in the particle size or the Zeta potential.

2. Nanoparticle freeze-drying stability

After the two kinds of liposomes were freeze-dried, ultrapure water was added again, and vortex stirring was carried out to make them sufficient. The results show that after sufficient time of vortexing, the nano bowl supported doxorubicin liposome group can be fully dispersed; however, the ordinary doxorubicin liposome component was not uniformly dispersed and in a suspension state, and a macroscopic red precipitate could be observed at the bottom of the tube after standing, as shown in fig. 15.a, D. Then, respectively sucking equivalent sample supernatants for DLS particle size detection, and taking an Intensity parameter as an investigation weight, wherein the result shows that the particle size distribution range of the common adriamycin liposome is remarkably widened and multimodal distribution is realized; in comparison, the doxorubicin liposome supported by the nanometer bowl still has unimodal distribution of particle size distribution, better dispersibility and particle size average value similar to that before freeze drying (fig. 15.B, C, E, F).

3. Storage stability of nanoparticles

The storage stability of the nanoparticles was monitored by periodically DLS measurements of particle size and Zeta potential on nanobowl-supported doxorubicin liposomes stored at 4 ℃. As shown in FIG. 16, the nanoparticles maintain stable particle size and Zeta potential for a period of 120 days, have good dispersibility, and no obvious sedimentation and agglomeration phenomena, thus proving that the nanoparticles can be stably stored for a long time.

4. 4T1 cell nanoparticle uptake

Under the magnification of the confocal laser microscope 40, the uptake of nanoparticles by 4T1 cells was observed with excitation light of the same intensity. As can be seen from fig. 17.a, the fluorescence intensity of doxorubicin in 4T1 cells gradually increased with the passage of time, and it is concluded that the uptake of drug-loaded nanoparticles by 4T1 cells also increased with the passage of time; meanwhile, after incubation for 4h, the fluorescence signal of the adriamycin in the nucleus area is obviously enhanced compared with that of the adriamycin in 1h, which prompts that the adriamycin begins to be released into the nucleus; however, regardless of the uptake time of 1h or 4h, the fluorescence intensity of the nano bowl-supported doxorubicin liposome and the fluorescence intensity of the common doxorubicin liposome in the cells are basically equal, no significant difference exists, and the fluorescence quantitative statistics are shown in fig. 17.B and are consistent with the observation phenomenon.

5. Effect of Nanoparticulates on cell viability

The adriamycin concentration range of 0.01-10.00 mu g/mL is selected as the investigation range of the research, and serum-free culture media containing nano-particle medicines or free medicines of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 mu g/mL are respectively prepared. And adding the liquid medicines into corresponding 96-well plates respectively, and incubating. And selecting the incubation time of 48h as an experimental test time point, and detecting the cell activity by a CCK-8 method. The results are shown in fig. 18.a, first, at the in vitro cell level, the effect of the free drug group on the cell viability is significantly stronger than that of the common adriamycin liposome and the nano bowl-supported adriamycin liposome; secondly, after incubation for 48h, the effect of the normal doxorubicin liposome group and the nano bowl-supported doxorubicin liposome group on cell viability was not statistically different. Therefore, the effect of the common adriamycin liposome group and the nano bowl-supported adriamycin liposome group on cell viability is basically equivalent on an in-vitro cell level, and compared with the two groups of liposomes, the free drug has stronger cytotoxicity. In addition, the nano bowl support liposome without drug loading is selected for CCK-8 detection, and is incubated for 48 hours with the concentration range of 0.01-3mg/mL, and no significant cell activity attenuation is seen, so that the biocompatibility of the nano particle is good, and the nano particle has no toxic effect on tumor cells even under the condition of higher dose.

Fourth, discuss

1. Selection of tumor cells

According to us FDA approved marketed Doxil and european approved marketed Myocet indication, doxorubicin liposomes are approved for the treatment of metastatic breast cancer with prolonged survival time and significantly reduced adverse reactions. The growth and transfer characteristics of the mouse 4T1 cell in BALB/c mouse are similar to those of breast cancer in human body. The tumor cell is widely used as an animal model of human VI-stage breast cancer. Therefore, this example selects 4T1 cells as the study object, and carries out the subsequent experiments.

2. Extracorporeal simulation circulation system

The circulatory system is the internal environment that is reached first after intravenous injection of the drug, in which the nano-drug will be affected by the interaction of various proteins, cytokines and cells and by the impact of the blood flow, thus producing a series of changes. However, because of the limitation of the conditions of small whole blood volume of the mouse, easy coagulation of the whole blood and the like, the environment of 100% fetal calf serum is selected, the interaction of the nano-drug with various proteins and cytokines and the impact of blood flow in the in vivo circulatory system is approximately simulated by constant temperature oscillation at 37 ℃, and the interaction with blood cells is not related at all.

3. Effect of Nano-bowl on stability

According to the results, the support of the nanometer bowl successfully reduces the drug leakage of the liposome in a serum environment and remarkably improves the redispersion capability of the nanometer preparation after freeze drying, in a circulatory system, the support effect of the nanometer bowl, namely ①, can offset the impact effect of partial blood flow on the liposome, reduce the deformation degree of the liposome, and further reduce the problems of liposome fracture and content leakage caused by excessive deformation, ②, the combination effect of various proteins and cytokines in serum and the liposome, and the change of liposome permeability is also another important reason for causing the leakage of liposome content, and the leakage range is greatly reduced due to the special opening structure of the nanometer bowl, and the leakage of the content is only caused when the phospholipid bilayer permeability in the bowl mouth is changed, so that the occurrence probability of the leakage is reduced.

4. Effect of the Nano-bowl liner on cytotoxicity and uptake Capacity

The problem of whether the addition of the nanometer bowl can lead the cells to have certain influence on the uptake capacity of the nanometer drugs or not is attracted by the research of the early design of the experiment. Cellular uptake of nanoparticles is generally related to the surface properties of the nanoparticles, such as nanoparticle morphology, surface groups, Zeta potential, and the like. Although the shape of the nanometer bowl is irregular, the liposome is completely coated, so that the surface of the liposome is completely covered by the phospholipid and still presents a vesicular shape. Therefore, the nanoparticle morphology, surface groups, Zeta potential are comparable to common liposomes. Confocal microscopy statistics also verify that the lining of the nanocowl does not affect uptake of liposomes by cells.

Similarly, whether the inner lining of the nano bowl influences the drug release behavior of the nano-drug, which has an influence on the cytotoxicity. With this doubt, the corresponding CCK-8 experiment was designed in this example. The results show that the toxicity of the drug-loaded liposome to the cells is not affected by the nano bowl. In addition, the blank nano bowl support liposome without drug loading also does not show obvious cytotoxicity, thus proving that the carrier has good biocompatibility.

Five, small knot

The stability of the nano-drug is comprehensively evaluated by investigating the changes of the particle size, the Zeta potential and the leakage rate of the nano-drug under three different environments and operations of a simulated circulation system, a freeze drying environment and a storage environment at 4 ℃. From the above experiments, it can be seen that the prepared nano bowl-supported doxorubicin liposome can maintain good dispersibility and extremely low leakage rate in the environments of the in vivo circulatory system, freeze drying and low-temperature storage. At the in vitro cell level, the uptake of the 4T1 cells to the nano-drug and the cytotoxicity of the nano-drug are examined. The results show that the support of the nanometer bowl has no significant effect on the cells, and the uptake of the nanometer bowl-supported adriamycin liposome by the cells is equivalent to that of the common adriamycin liposome and the toxicity level of the liposome to the cells.

Example 4 in vivo evaluation of Nanowbowl Supported Doxorubicin liposomes for antitumor therapy

Instrument and material

1. Instrumentation and equipment

Watch 13

Model XS205s electronic balance	Mettlerlington Co Ltd
		JY 92-II ultrasonic cell crusher	NINGBO SCIENTZ BIOTECHNOLOGY Co.,Ltd.
HS-70 type constant temperature magnetic stirrer	IKA, Germany
		R-200 rotary evaporator	Buchi Switzerland
Vacuum drying oven	Shanghai-constant technology Instrument Co Ltd
		Mini extrusion device	Avanti corporation of America
SorvallST16 refrigerated centrifuge	Sammerfo USA SA
		CM-120 transmission electron microscope	Philips, Netherlands
Malvern Zetasizer NanoZS laser particle size analyzer	Malvern, UK
		SpectraMaxM2 biomolecule enzyme-labeling instrument	Molecular instruments, Inc. of USA
ZQTY-70 shaking incubator	SHANGHAI ZHICHU INSTRUMENT Co.,Ltd.
		MS2 vortex mixer	IKA, Germany
Laboratory ultrapure water system	Millipore Inc. USA
		LSM5 laser confocal microscope	Zeiss in Germany
DP50 upright microscope	Olympus corporation of Japan
		Carbon dioxide cell incubator	Sammerfo USA SA
Invitrogen enlifeficocontess cell counter	Sammerfo USA SA
		1300SeriesA2 superclean operation platform	Sammerfo USA SA
Olympus electron microscope	Olympus corporation of Japan

2. Materials and reagents

TABLE 14

3. Cell line and animal

4T1 Breast cancer cells, purchased from Caliper Life Sciences, Inc. (Hopkinton, Mass.). BALB/c female mice, 4-6 weeks old, were supplied by Shanghai Spiker laboratory animals, Inc.

4. Preparation of related reagents

2) 4% paraformaldehyde solution: taking PBS (0.01M) with pH 7.4 as a solvent, weighing 40g of paraformaldehyde, putting into 800ml PBS solution, oscillating and dissolving overnight in a constant-temperature shaking table at 60 ℃, stopping heating after the paraformaldehyde solid is completely dissolved, balancing to room temperature, and adding PBS again to fix the volume to 1L;

3) 1% pentobarbital sodium anesthetic: 100mg of sodium pentobarbital was weighed, added to 10mL of ultrapure water, and vortexed until fully dissolved.

Second, Experimental methods

1. Establishment of 4T1 breast cancer orthotopic inoculation model

1.14T 1 cell suspension preparation

1) Taking a bottle of T-75 culture flask which is used for culturing 4T1 cells, sucking the cell culture solution in the culture flask by using a pipette, adding 5mL of DPBS (double DPBS) for rinsing for 1-2 times to remove the residual culture solution;

2) adding 2mL of 0.25% trypsin, shaking the flask back and forth to allow the trypsin to cover the entire bottom surface of the flask, placing at 37 deg.C and containing 5% CO₂Incubating for 3min in the constant temperature incubator;

3) adding 10mL of serum-free cell culture medium, diluting pancreatin, and stopping digestion;

4) blowing the cell suspension by using a pipettor to uniformly disperse the cell suspension to obtain single cell suspension, taking 5 mu L of the cell suspension, adding the same volume of placenta blue staining solution, and counting cells of the cell suspension by using an Invitrogen life countess cell counter;

5) centrifuging at 4 deg.C for 5min at 800g, discarding supernatant, calculating intracellular concentration,adding a proper amount of serum-free cell culture medium, and gently blowing and beating the cell blocks by using a pipettor to fully disperse the cell blocks to prepare single cell suspension, wherein the final concentration of the cell suspension is as follows: 1.6X 10⁷Per mL;

6) the 4T1 cell suspension was dispensed into sterile 2mL centrifuge tubes and refrigerated at 4 ℃ until use.

1.2BALB/c female mice were inoculated orthotopically with 4T1 cells

1) The right abdominal hair of the mouse is removed the day before inoculation, and skin preparation is made for the next day of operation;

2) the next day, the mice treated with the preserved skin were injected intraperitoneally with 100 μ L of 1% pentobarbital anesthetic;

3) fixing limbs after the mice are completely anesthetized, wetting the right abdomen with physiological saline, and slightly stroking one mouth on the abdomen with a scalpel;

4) the 4 th mammary fat pad at the root of the right thigh was clamped with forceps and 50. mu.l of the 4T1 single cell suspension prepared previously was injected;

5) loosening the forceps, returning the fat pad to the original position, and suturing the abdominal wound surface;

6) after inoculation, mice were returned to their cages and the front legs of each mouse were attached to a label strip.

2. Animal grouping and dosing regimens

1)4 days after inoculation, tumor volume was measured with a vernier caliper: the longest and shortest tumor diameters (L, W) were measured every 1 day and tumor volumes were calculated according to the following formula:

2) when the tumor volume grows to 80mm³Then, based on the tumor volume size, the following 5 experimental groups were evenly divided:

① Saline group (Saline)

② blank nanometer bowl support liposome group (NB @ LP)

③ doxorubicin hydrochloride group (Dox)

④ common Doxorubicin liposome group (DLP)

⑤ nanometer bowl supporting doxorubicin liposome group (NB @ DLP)

3) On 8, 11 and 14 days after inoculation, the mice in each experimental group were injected with the same amount of the corresponding drug in the tail vein, and each drug-containing group was administered with 4mg/kg of doxorubicin.

3. Mouse weight and tumor volume monitoring

1) After the first needle administration in groups, the body weight and the length and the diameter of the tumor of the mouse are measured once every 2 days, the volume of the tumor is calculated, and all results are recorded and summarized;

2) data induction and collation, statistical analysis and charting were performed using GraphPad prism7.0 medical mapping software.

4. Immunohistochemical staining of tissue sections

1) Selecting a plurality of mice in each experimental group after the last intravenous administration, dissecting and picking tumor tissues and important organs of the mice after the mice are killed, and fully soaking the mice in 4% paraformaldehyde solution for fixation;

2) during the fixed period, change fresh 4% paraformaldehyde solution many times to wash away residual blood, treat that the supernatant solution clarifies the back, paraffin embedding, section, immunohistochemical staining: TUNEL, PCNA and H & E staining;

3) and observing the stained section under a microscope, taking a picture, collecting an Image, analyzing the Image by using Image-Pro Plus 6.0 software, and calculating the apoptosis positive rate and the proliferation positive rate of the tumor cells.

5. Observation of mouse survival

1) After the administration is finished, the body weight and the tumor length and the tumor diameter of the mice are measured once every 2 days;

2) observing the mental state, vital signs and survival state of the mouse;

3) when the mice die or the tumor-bearing volume of the mice exceeds 2000mm³At that time, death was recorded while euthanizing the mice.

6. Statistical treatment

Three, result in

1. Pharmacodynamic evaluation of nano bowl-supported adriamycin liposome for treating breast cancer

To examine the therapeutic effect of the nano bowl-supported doxorubicin liposome on 4T1 breast cancer, BALB/c female mice inoculated with tumor cells in the normal position were administered after one week, and the corresponding drugs were administered by tail vein injection on

days

0, 3 and 6, respectively. Mouse body weight, tumor volume and mouse survival were recorded from the first dose (day 0) and observed, and survival curves were plotted, with the results shown in fig. 19. As can be seen from the graphs in FIG. 19 and B, the tumor volume increase rates of the free adriamycin, the common adriamycin liposome and the nano-bowl-supported adriamycin liposome are all slowed down to different degrees compared with the control group, which indicates that the adriamycin can effectively inhibit the tumor growth. However, compared with three groups of drug-containing use experimental groups, the drug-containing use experimental groups can find that the nano bowl-supported adriamycin lipid has the most obvious curative effect and almost no growth of tumors according to the order of the tumor inhibition effect from good to poor; the second is the common adriamycin liposome, although the common adriamycin liposome can not completely inhibit the growth of the tumor like the nano bowl supported adriamycin liposome group, the overall growth rate is slow; the worst is the free adriamycin administration group, although the volume is increased slowly, the free adriamycin administration group is far from reaching the expectation of treating the tumor, and the reason is probably related to the rapid in vivo clearance rate of the free adriamycin, so that the free adriamycin cannot be effectively accumulated at the tumor part. In addition, by monitoring the weight of the mice (fig. 19.C), the free adriamycin administration group was found to have a significant weight loss, a slimy body and a certain deterioration of mental status during the administration period, and it was found that the free adriamycin itself has a certain degree of toxic and side effects on the body.

Similarly, fig. 19.D and table 15 show the survival curves of mice in the saline group, the empty nanobowl-supported liposome, free doxorubicin, ordinary doxorubicin liposome and nanobowl-supported doxorubicin liposome groups, which have median survival periods of 24 days, 27 days, 30 days and 50 days, respectively. Compared with a normal saline control group, the drug-containing administration groups prolong the life cycle of the tumor-bearing mice to different degrees; wherein, compared with a control group, the free adriamycin has little curative effect, and the survival time of the Zhongwei is only prolonged by 3 days (12.50%); compared with the control group, the common adriamycin liposome prolongs the treatment period by 6 days (25.00%), and the curative effect is improved compared with the free adriamycin; in three groups of administration groups, the nano bowl supported adriamycin liposome has the most remarkable curative effect, the median survival time is prolonged to the longest extent, the median survival time is prolonged by 50 days, the survival time is prolonged by 26 days (108.33%) compared with a control group, and the survival time is prolonged by 20 days (66.67%) compared with the common adriamycin liposome. In addition, 1 death was not observed within 72 days of observation.

TABLE 15 median survival of 4T1 breast cancer mice after administration to different experimental groups

2. Nano medicine for promoting tumor apoptosis and inhibiting tumor proliferation

According to the inhibition effect of each experimental group on the 4T1 tumor, tumor tissues of tumor-bearing mice are picked up, pathological sections and immunohistochemical staining analysis are carried out, TUNEL and PCNA staining are respectively carried out, and the apoptosis and proliferation conditions of tumor cells are detected. The results are shown in fig. 20.a, the drug-containing experimental group has different degrees of inhibition on apoptosis of tumor cells, and simultaneously inhibits proliferation of tumor cells to different degrees; wherein, the nano bowl supported adriamycin liposome has the most obvious effects of promoting apoptosis and inhibiting proliferation, the common adriamycin liposome has the second effect, the free adriamycin has the worst effect, and the semi-quantitative result is shown in figures 20.B and C; the blank nano-carrier group has no obvious curative effect and is consistent with the results of in vitro cell experiments.

3. Toxicity analysis of nano-drug on important organs

Similarly, the important organ tissues of tumor-bearing mice were picked up, stained with pathological sections, and observed to examine the effects of different administration groups on the mouse organs. The results are shown in fig. 21, and each experimental group did not cause significant toxicity to the liver, kidney, spleen and lung of the mice; free adriamycin has high toxicity to heart, so that myocardial cells are dissolved and broken; the adriamycin liposome supported by the nanometer bowl and the common adriamycin liposome can obviously reduce the toxic and side effect of adriamycin on the heart, and pathological sections have no obvious pathological changes; in addition, the blank nanoparticle carrier group has no obvious toxicity on tissues such as heart, liver, spleen, lung, kidney and the like, and the carrier is non-toxic and has good biocompatibility.

Fourth, discuss

1. Nano bowl support for overall treatment effect on in-vivo breast cancer

According to the experimental results, the support of the nanometer bowl can show the curative effect which is obviously better than that of other experimental groups in the treatment of the breast cancer of the mouse, and the effect of inhibiting the tumor volume and prolonging the survival curve of the mouse are obviously improved compared with that of other groups, and the result is exactly opposite to that of the in vitro cell level in example 3. At the cellular level in vitro, the most cytotoxic free doxorubicin is the least effective; the tumor inhibition effect of the common adriamycin liposome originally in vitro and not classified into primary and secondary with the nano bowl-supported adriamycin liposome in a mouse body is not as prominent as that of the nano bowl-supported adriamycin liposome, but still has a little better than that of free adriamycin; the nano bowl supported adriamycin liposome shows the best tumor inhibition effect, and the tumor volume is not increased any more. Firstly, the extremely poor long-circulating characteristic and the extremely fast clearance rate of the free adriamycin cause that a large amount of adriamycin can not really reach the tumor part, thereby influencing the curative effect of the adriamycin; in the case of the adriamycin liposome, although the entrapment of the liposome can increase the retention time of the adriamycin liposome in a circulatory system to a certain extent, experiments prove that the phenomenon of early leakage of the drug still occurs, so that the amount of the drug reaching a tumor part is reduced to a certain extent; the supporting function of the nanometer bowl provides a hard inner container for the liposome, so that the liposome can bear the impact and damage of various factors in a circulatory system, the leakage of content drugs is reduced as far as possible before reaching a tumor part, more adriamycin reaches the tumor part along the circulatory system, and the curative effect of the drug is better exerted.

2. Influence of nanometer bowl support on tumor and important visceral organs

The research on mouse tumor tissues and important organ tissue slices shows that the nano bowl-supported adriamycin liposome can better inhibit tumor cell proliferation, promote tumor cell apoptosis and reduce the toxic and side effects of adriamycin on the heart compared with the common adriamycin liposome. The reason may be closely related to the supporting function of the nanometer bowl. The supporting effect of the nanometer bowl can reduce the early leakage of the adriamycin in the in vivo circulation process, so that the adriamycin can carry more drugs to reach the tumor part, thereby improving the curative effect; meanwhile, the concentration of the drug in the organ tissues is reduced due to the reduction of the leakage of the free drug, so that the toxicity to other organs is reduced.

Five, small knot

In the method, a 4T1 breast cancer model is successfully constructed and inoculated in a positive position on a BALB/c female mouse, different medicines are injected into the tail vein of a tumor-bearing mouse, and the treatment effect of each group of medicines is observed. The macroscopic curative effect of the mice after administration is integrally evaluated by detecting the weight, the tumor volume and the survival curve of the mice; by immunohistochemical staining analysis of tumor tissues and important organs, the apoptosis, proliferation and necrosis of tumor tissues are examined, and the anti-tumor treatment effect is further analyzed and evaluated from a microscopic angle. The results show that the nano bowl supported adriamycin liposome has the best overall anti-tumor effect, and is the common adriamycin liposome, while the free adriamycin group has poor curative effect, the body weight of a mouse has a remarkable reduction trend during administration, and the H & E results also show that the nano bowl supported adriamycin liposome has high toxicity to the heart.

Example 5 preparation of Nano bowl-Supported irinotecan/vincristine liposomes

The preparation method is basically the same as that of example 1-2, and reference is made to example 1-2, except that adriamycin is replaced by irinotecan or vincristine.

Example 6 Effect experiment of Nano bowl-supported irinotecan/vincristine liposome

The first experiment method comprises the following steps:

to detect serum-induced membrane instability and Irinotecan or Vincristine leakage, Irinotecan (Irinotecan) or Vincristine (Vincristine) liposomes or nanobowl-supported liposomes were dispersed in pure FBS (fetal bovine serum) at 37 deg.C, shaken at 200rpm, and after a certain time, the liposomes were purified using Zebaspin desalting column (Thermo-Scientific) to remove Irinotecan or Vincristine outside the liposomes. The drug-retaining liposomes were mixed with 9 volumes of 0.75M HCl (90% isopropanol) and centrifuged. The supernatant containing irinotecan or vincristine was evaporated in a SpeedVac concentrator (Thermo Scientific) and the residue dissolved in 200 μ l of mobile phase consisting of methanol, acetonitrile, water and trifluoroacetic acid at 24:24:52:0.1(v/v, pH 3.0) at a flow rate of 1mL/min, separated on a diamonsil C18 column (4.6 x 150m m, 5 μm, Dikma, China) and detected at 254 nm. Leakage of liposomes in FBS was calculated.

Second, experimental results

The results show that compared with the common Irinotecan liposome (Irinotecan-LP) and the Vincristine liposome (Vincristine-LP), the nano-bowl supported Irinotecan liposome (NB @ Irinotecan-LP) and the Vincristine liposome (NB @ Vincristine-LP) have remarkably reduced leakage rate and show high serum stability.

The invention successfully synthesizes the nano particles with the bowl-shaped structure, perfects and optimizes the synthesis prescription of the nano particles, and establishes a related characterization and identification method of a whole set of nano bowls. Successfully constructs a nano bowl-supported drug-loaded liposome drug delivery system, explores and establishes a loading mode of adriamycin hydrochloride/irinotecan/vincristine, and realizes high encapsulation rate of adriamycin/irinotecan/vincristine. Subsequently, the invention comprehensively discusses various stabilities of the nanometer bowl supported drug-loaded liposome, and evaluates the cell uptake behavior and cytotoxicity of the nanometer bowl supported drug-loaded liposome at the in vitro cell level. The stability test result shows that the nano bowl supported drug-loaded liposome can reduce the leakage rate of the drug-loaded liposome in a circulatory system; meanwhile, the freeze-dried powder can be better redispersed after freeze-drying treatment, so that the freeze-dried powder can be conveniently prepared; in addition, the nano bowl-supported adriamycin liposome dispersion can be stored for 120 days in an environment of 4 ℃, and has no sedimentation or agglomeration phenomenon and good dispersibility. Cell test results show that the addition of the nano bowl does not negatively influence the cell uptake behavior and cytotoxicity of the drug-loaded liposome. Finally, the invention successfully constructs a 4T1 breast cancer orthotopic inoculation model, and inspects the treatment effect of the nano bowl-supported adriamycin liposome on breast cancer. The results show that the doxorubicin liposome supported by the nano bowl can effectively inhibit the growth of tumors and remarkably prolong the life cycle of tumor-bearing mice; meanwhile, compared with other four experimental groups, the doxorubicin liposome supported by the nano bowl can obviously reduce the proliferation of tumor cells and promote the apoptosis of the tumor cells; the body weight and organ staining results of the mice show that the doxorubicin liposome supported by the nano bowl can effectively reduce the toxic and side effects of the doxorubicin and improve the survival quality of the mice. The invention discloses a novel method for improving the anti-tumor curative effect by improving the circulation stability of liposome and reducing the leakage of a medicament, and provides a novel thought and theoretical basis for anti-tumor treatment.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

Claims

1. The nanometer bowl supported drug-loaded liposome is characterized in that the preparation method comprises the following steps:

2. The nano bowl supported drug-loaded liposome of claim 1, wherein the drug in step (3) is selected from any one of adriamycin, irinotecan and vincristine.

3. The preparation method of the nano bowl-supported drug-loaded liposome as claimed in claim 1, which comprises the following steps:

4. The nano bowl supported adriamycin/irinotecan/vincristine liposome is characterized by comprising the following steps:

5. The nano-bowl supported doxorubicin/irinotecan/vincristine liposome according to claim 4, wherein the polystyrene nanoparticles in step (1) have a particle size of 45-55 nm; the peanut-shaped nano particles are polystyrene nano particles modified by styrene monomers and MPS according to V_styrene:V_MPSNPsPrepared as 3: 1; the silica-modified peanut-like nanoparticles were prepared with 0.3g TEOS.

6. The nano-bowl supported adriamycin/irinotecan/vincristine liposome according to claim 4, wherein the nano-bowl supported adriamycin/irinotecan/vincristine liposome has an average particle size of 140-150nm and a Zeta potential of-18 to-16 mV.

7. The method for preparing the nano bowl supported adriamycin/irinotecan/vincristine liposome of claim 4, which is characterized by comprising the following steps: (1) preparing a nanometer bowl: preparing polystyrene nano-particles → preparing MPS coated modified polystyrene nano-particles → preparing peanut-shaped nano-particles → preparing silica modified peanut-shaped nano-particles → obtaining a nano bowl;

8. The preparation method of claim 7, wherein the preparation method of the nano bowl in the step (1) comprises the following steps:

9. Preparation according to claim 7The method is characterized in that the particle size of the polystyrene nano-particles in the step (1) is 50 nm; the peanut-shaped nano particles are polystyrene nano particles modified by styrene monomers and MPS according to V_styrene:V_MPSNPsPrepared as 3: 1; the silica-modified peanut-like nanoparticles were prepared with 0.3g TEOS.

10. The use of the nano bowl supported drug-loaded liposome of claim 1 in the preparation of an anti-tumor medicament.